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Chlamydia trachomatis serovar L2 and Chlamydia muridarum, which do not express FtsZ, undergo polarized cell division. During division, peptidoglycan assembles at the pole of dividing Chlamydia trachomatis cells where daughter cell formation occurs, and peptidoglycan regulates at least two distinct steps in the polarized division of Chlamydia trachomatis and Chlamydia muridarum. Cells treated with inhibitors that prevent peptidoglycan synthesis or peptidoglycan crosslinking by penicillin-binding protein 2 (PBP2) are unable to initiate polarized division, while cells treated with inhibitors that prevent peptidoglycan crosslinking by penicillin-binding protein 3 (PBP3/FtsI) initiate polarized division, but the process arrests at an early stage of daughter cell growth. Consistent with their distinct roles in polarized division, peptidoglycan organization is different in cells treated with PBP2 and PBP3-specific inhibitors. Our analyses indicate that the sequential action of PBP2 and PBP3 drives changes in peptidoglycan organization that are essential for the polarized division of these obligate intracellular bacteria. Furthermore, the roles we have characterized for PBP2 and PBP3 in regulating specific steps in chlamydial cell division have not been described in other bacteria.

Chlamydiae lack the conserved central coordinator protein of cell division FtsZ, a tubulin-like homolog. Current evidence indicates that Chlamydia uses the actin-like homolog, MreB, to substitute for the role of FtsZ in a polarized division mechanism. Interestingly, we observed MreB as a ring at the septum in dividing cells of Chlamydia We hypothesize that MreB, to substitute for FtsZ in Chlamydia , must possess unique properties compared to canonical MreB orthologs. Sequence differences between chlamydial MreB and orthologs in other bacteria revealed that chlamydial MreB possesses an extended N-terminal region, harboring predicted amphipathicity, as well as the conserved amphipathic helix found in other bacterial MreBs. The conserved amphipathic helix-directed green fluorescent protein (GFP) to label the membrane uniformly in Escherichia coli but the extended N-terminal region did not. However, the extended N-terminal region together with the conserved amphipathic region directed GFP to restrict the membrane label to the cell poles. In Chlamydia , the extended N-terminal region was sufficient to direct GFP to the membrane, and this localization was independent of an association with endogenous MreB. Importantly, mutating the extended N-terminal region to reduce its amphipathicity resulted in the accumulation of GFP in the cytosol of the chlamydiae and not in the membrane. The N-terminal domain of MreB was not required for homotypic interactions but was necessary for interactions with cell division components RodZ and FtsK. Our data provide mechanistic support for chlamydial MreB to serve as a substitute for FtsZ by forming a ringlike structure at the site of polarized division. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen, causing sexually transmitted diseases and trachoma. The study of chlamydial physiology is important for developing novel therapeutic strategies for these diseases. Chlamydiae divide by a unique MreB-dependent polarized cell division process. In this study, we investigated unique properties of chlamydial MreB and observed that chlamydial species harbor an extended N-terminal region possessing amphipathicity. MreB formed a ring at the septum, like FtsZ in Escherichia coli , and its localization was dependent upon the amphipathic nature of its extended N terminus. Furthermore, this region is crucial for the interaction of MreB with cell division proteins. Given these results, chlamydial MreB likely functions at the septum as a scaffold for divisome proteins to regulate cell division in this organism., (Copyright © 2020 American Society for Microbiology.)

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections, and Chlamydia pneumoniae causes community-acquired respiratory infections. In vivo , the host immune system will release gamma interferon (IFN-γ) to combat infection. IFN-γ activates human cells to produce the tryptophan (Trp)-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Consequently, there is a reduction in cytosolic Trp in IFN-γ-activated host cells. In evolving to obligate intracellular dependence, Chlamydia has significantly reduced its genome size and content, as it relies on the host cell for various nutrients. Importantly, C. trachomatis and C. pneumoniae are Trp auxotrophs and are starved for this essential nutrient when the human host cell is exposed to IFN-γ. To survive this, chlamydiae enter an alternative developmental state referred to as persistence. Chlamydial persistence is characterized by a halt in the division cycle, aberrant morphology, and, in the case of IFN-γ-induced persistence, Trp codon-dependent changes in transcription. We hypothesize that these changes in transcription are dependent on the particular amino acid starvation state. To investigate the chlamydial response mechanisms acting when other amino acids become limiting, we tested the efficacy of prokaryote-specific tRNA synthetase inhibitors, indolmycin and AN3365, to mimic starvation of Trp and leucine, respectively. We show that these drugs block chlamydial growth and induce changes in morphology and transcription consistent with persistence. Importantly, growth inhibition was reversed when the compounds were removed from the medium. With these data, we find that indolmycin and AN3365 are valid tools that can be used to mimic the persistent state independently of IFN-γ., (Copyright © 2020 American Society for Microbiology.)

Expansion microscopy (ExM) is a novel tool to improve the resolution of fluorescence-based microscopy that has not yet been used to visualize intracellular pathogens. Here we show the expansion of the intracellular pathogen Chlamydia trachomatis , enabling to differentiate its two distinct forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We show that ExM enables the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside of the chlamydial inclusion. Thus, we claim that ExM offers the possibility to address a broad range of questions and may be useful for further research on various intracellular pathogens.

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection. It produces an unusual intracellular infection in which a vegetative form, called the reticulate body (RB), replicates and then converts into an elementary body (EB), which is the infectious form. Here we use quantitative three-dimensional electron microscopy (3D EM) to show that C. trachomatis RBs divide by binary fission and undergo a sixfold reduction in size as the population expands. Conversion only occurs after at least six rounds of replication, and correlates with smaller RB size. These results suggest that RBs only convert into EBs below a size threshold, reached by repeatedly dividing before doubling in size. A stochastic mathematical model shows how replication-dependent RB size reduction produces delayed and asynchronous conversion, which are hallmarks of the Chlamydia developmental cycle. Our findings support a model in which RB size controls the timing of RB-to-EB conversion without the need for an external signal.

The lifestyle of Chlamydiae is unique: the bacteria alternate between two morphologically distinct forms, an infectious non-replicative elementary body (EB), and a replicative, non-infectious reticulate body (RB). This review focuses on recent advances in understanding the structure and function of the infectious form of the best-studied member of the phylum, the human pathogen Chlamydia trachomatis. Once considered as an inert particle of little functional capacity, the EB is now perceived as a sophisticated entity that encounters at least three different environments during each infectious cycle. We review current knowledge on its composition and morphology, and emerging metabolic activities. These features confer resistance to the extracellular environment, the ability to penetrate a host cell and ultimately enable the EB to establish a niche enabling bacterial survival and growth. The bacterial and host molecules involved in these processes are beginning to emerge.

Coxiella burnetii is an obligate intracellular pathogen and the causative agent of human Q fever. Replication of the bacterium within a large parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. PV biogenesis is a pathogen driven process that requires engagement of several host cell vesicular trafficking pathways to acquire vacuole components. The goal of this study was to determine if infection by C . burnetii modulates endolysosomal flux to potentially benefit PV formation. HeLa cells, infected with C. burnetii or left uninfected, were incubated with fluorescent transferrin (Tf) for 0-30 min, and the amount of Tf internalized by cells quantitated by high-content imaging. At 3 and 5 days, but not 1 day post-infection, the maximal amounts of fluorescent Tf internalized by infected cells were significantly greater than uninfected cells. The rates of Tf uptake and recycling were the same for infected and uninfected cells; however, residual Tf persisted in EEA.1 positive compartments adjacent to large PV after 30 min of recycling in the absence of labeled Tf. On average, C. burnetii -infected cells contained significantly more CD63-positive endosomes than uninfected cells. In contrast, cells containing large vacuoles generated by Chlamydia trachomatis exhibited increased rates of Tf internalization without increased CD63 expression. Our results suggest that C. burnetii infection expands the endosomal system to increase capacity for endocytic material. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to infection by intracellular pathogens.

Cell division in Chlamydiae is poorly understood as apparent homologs to most conserved bacterial cell division proteins are lacking and presence of elongation (rod shape) associated proteins indicate non-canonical mechanisms may be employed. The rod-shape determining protein MreB has been proposed as playing a unique role in chlamydial cell division. In other organisms, MreB is part of an elongation complex that requires RodZ for proper function. A recent study reported that the protein encoded by ORF CT009 interacts with MreB despite low sequence similarity to RodZ. The studies herein expand on those observations through protein structure, mutagenesis and cellular localization analyses. Structural analysis indicated that CT009 shares high level of structural similarity to RodZ, revealing the conserved orientation of two residues critical for MreB interaction. Substitutions eliminated MreB protein interaction and partial complementation provided by CT009 in RodZ deficient Escherichia coli. Cellular localization analysis of CT009 showed uniform membrane staining in Chlamydia. This was in contrast to the localization of MreB, which was restricted to predicted septal planes. MreB localization to septal planes provides direct experimental observation for the role of MreB in cell division and supports the hypothesis that it serves as a functional replacement for FtsZ in Chlamydia., (© 2014 John Wiley & Sons Ltd.)

Chlamydia trachomatis is an obligate intracellular pathogen responsible for a high burden of human disease. Here, a loss-of-function screen using a set of lentivirally transduced shRNAs identified 14 human host cell factors that modulate C. trachomatis infectivity. Notably, knockdown of dynamin, a host GTPase, decreased C. trachomatis infectivity. Dynamin functions in multiple cytoplasmic locations, including vesicle formation at the plasma membrane and the trans-Golgi network. However, its role in C. trachomatis infection remains unclear. Here we report that dynamin is essential for homotypic fusion of C. trachomatis inclusions but not for C. trachomatis internalization into the host cell. Further, dynamin activity is necessary for lipid transport into C. trachomatis inclusions and for normal re-differentiation from reticulate to elementary bodies. Fragmentation of the Golgi apparatus is proposed to be an important strategy used by C. trachomatis for efficient lipid acquisition and replication within the host. Here we show that a subset of C. trachomatis-infected cells displayed Golgi fragmentation, which was concurrent with increased mitotic accumulation. Golgi fragmentation was dispensable for dynamin-mediated lipid acquisition into C. trachomatis inclusions, irrespective of the cell cycle phase. Thus, our study reveals a critical role of dynamin in host-derived lipid acquisition for C. trachomatis development., (© 2014 John Wiley & Sons Ltd.)

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

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 pressure. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis and exhibit susceptibility to 'anti-PG' antibiotics, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the 'chlamydial anomaly'). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are 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 so far that chlamydial species possess functional PG.

Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.

Chlamydiae are obligate intracellular bacterial pathogens that have extensively reduced their genome in adapting to the intracellular environment. The chlamydial genome contains only three annotated cell division genes and lacks ftsZ. How this obligate intracellular pathogen divides is uncharacterized. Chlamydiae contain two high-molecular-weight (HMW) penicillin binding proteins (Pbp) implicated in peptidoglycan synthesis, Pbp2 and Pbp3/FtsI. We show here, using HMW Pbp-specific penicillin derivatives, that both Pbp2 and Pbp3 are essential for chlamydial cell division. Ultrastructural analyses of antibiotic-treated cultures revealed distinct phenotypes: Pbp2 inhibition induced internal cell bodies within a single outer membrane whereas Pbp3 inhibition induced elongated phenotypes with little internal division. Each HMW Pbp interacts with the Chlamydia cell division protein FtsK. Chlamydiae are coccoid yet contain MreB, a rod shape-determining protein linked to Pbp2 in bacilli. Using MreB-specific antibiotics, we show that MreB is essential for chlamydial growth and division. Importantly, co-treatment with MreB-specific and Pbp-specific antibiotics resulted in the MreB-inhibited phenotype, placing MreB upstream of Pbp function in chlamydial cell division. Finally, we showed that MreB also interacts with FtsK. We propose that, in Chlamydia, MreB acts as a central co-ordinator at the division site to substitute for the lack of FtsZ in this bacterium., (© 2012 Blackwell Publishing Ltd.)

Background: Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment., Results: Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response., Conclusion: Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are the main lipid components of bacterial outer membranes and are essential for cell viability in most Gram-negative bacteria. Here we show that small molecule inhibitors of LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase], the enzyme that catalyzes the first committed step in the biosynthesis of lipid A, block the synthesis of LOS in the obligate intracellular bacterial pathogen Chlamydia trachomatis. In the absence of LOS, Chlamydia remains viable and establishes a pathogenic vacuole ("inclusion") that supports robust bacterial replication. However, bacteria grown under these conditions were no longer infectious. In the presence of LpxC inhibitors, replicative reticulate bodies accumulated in enlarged inclusions but failed to express selected late-stage proteins and transition to elementary bodies, a Chlamydia developmental form that is required for invasion of mammalian cells. These findings suggest the presence of an outer membrane quality control system that regulates Chlamydia developmental transition to infectious elementary bodies and highlights the potential application of LpxC inhibitors as unique class of antichlamydial agents.

Chlamydiae growing in target mucosal human epithelial cells in vitro can transition from their normal developmental cycle progression, alternating between infectious but metabolically inactive elementary bodies to metabolically active but noninfectious reticulate bodies (RBs) and back to elementary bodies, into a state of persistence. Persistence in vitro is defined as viable but noncultivable chlamydiae involving morphologically enlarged, aberrant, and nondividing RBs. The condition is reversible, yielding infectious elementary bodies after removal of the inducers, including penicillin, interferon-gamma, iron or nutrient starvation, concomitant herpes infection, or maturation of the host cell into its physiologically differentiated state. All aberrant RB phenotypes are not the same, owing to differing up- or down-regulated chlamydial gene sets and subsequent host responses. Although all persistence-inducing conditions exist in vivo, key questions include (1) whether or not aberrant chlamydial RBs occur in vivo during the alternating acute-silent chronic-acute chlamydial infection scenario that exists in infected patients and animals and (2) whether such aberrant RBs can contribute to prolonged, chronic inflammation, fibrosis, and scarring.

Objective: To evaluate the efficiency of sperm washing procedures to remove Chlamydia trachomatis from semen both in clinical samples and experimental inoculations., Design: Laboratory-based study., Setting: Research laboratory in a university hospital., Patient(s): One hundred men attending for diagnostic semen analysis as part of infertility investigations and three sperm donors providing ejaculates for research purposes., Main Outcome Measure(s): Number of DNA copies of C. trachomatis, infectivity in an HeLa cell monolayer, and immunofluorescence., Result(s): Of the 100 semen samples examined, 13 contained detectable levels of C. trachomatis DNA (675-15,920 copies/mL) and in only 7 was this completely removed after sperm washing. In the remaining six DNA-positive samples, the number of copies in the postwash preparation ranged from 36-455 per mL. Experimental inoculations found that postwash preparations containing C. trachomatis DNA as low as 61 copies/mL were able to establish an infection in vitro., Conclusion(s): Undiagnosed C. trachomatis infections in men attending for assisted conception could potentially lead to infection or contamination of the IVF culture system as sperm washing methods are not 100% effective.

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.

Chlamydia trachomatis possesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless human C. trachomatis strain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic of C. trachomatis strains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 x 10(6) inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 x 10(4) IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes was glgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmid's ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

Chlamydia trachomatis and C. muridarum, human and mouse pathogens, respectively, share more than 99% of open reading frames (ORFs) but differ in a cytotoxin locus. Presence or absence of cytotoxin gene(s) in these strains correlates with their ability to grow in IFN-gamma treated mouse cells. Growth of toxin-positive C. muridarum is not affected in IFN-gamma treated cells, whereas growth of toxin-negative C. trachomatis is inhibited. We previously reported that this difference in IFN-gamma sensitivity is important to the in vivo infection tropism of these pathogens. Here we describe a phenotypic rescue assay that utilizes C. muridarum gamma irradiated killed elementary bodies (iEB) to rescue C. trachomatis infectivity in IFN-gamma treated mouse cells. Rescue by iEB was temporal, maximal early post infection, directly related to multiplicity of iEB infection, and was independent of de novo chlamydial transcription. Lastly, C. muridarum iEB vacuoles and C. trachomatis inclusions were not fusogenic, suggesting the factor(s) responsible for rescue was secreted or exposed to the cytosol where it inactivated IFN-gamma induced effectors. Chlamydial phenotypic rescue may have broader utility for the study of other EB associated virulence factors that function early in the interaction of chlamydiae with host cells.

Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for approximately 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti-C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.

Intracellular parasitism by Chlamydiales is a complex process involving transmission of metabolically inactive particles that differentiate, replicate, and re-differentiate within the host cell. A type three secretion system (T3SS) has been implicated in this process. We have here identified small molecules of a chemical class of acylated hydrazones of salicylaldehydes that specifically blocks the T3SS of Chlamydia. These compounds also affect the developmental cycle showing that the T3SS has a pivotal role in the pathogenesis of Chlamydia. Our results suggest a previously unexplored avenue for development of novel anti-chlamydial drugs.

Background: A large amount (80,000-100,000 pg/ml) of interleukin-6 (IL-6) was detected in cultures of human synoviocytes infected with Chlamydia trachomatis. In this study, we investigated the effect of antibiotics on the IL-6 production of C. trachomatis-infected human fibroblast-like synovial cells (HFLS)., Methods: The minimum inhibitory concentrations of levofloxacin and doxycycline against C. trachomatis were determined in either HFLS or HeLa 229 cells. The number of live Chlamydia was also examined. IL-6 in the supernatants of infected cultures was quantified by capture ELISA., Results: The production of IL-6 was suppressed to as low as 1,800 pg/ml in the infected HFLS treated with levofloxacin or doxycycline immediately or early after infection. In HFLS treated with levofloxacin and doxycycline, the IL-6 levels decreased to 37,000 and 21,000 pg/ml, respectively, 48 h after infection, and levofloxacin was thus found to be less effective than doxycycline. In addition, the number of viable C. trachomatis in the infected cultures treated with levofloxacin 24 h after infection was higher than when treated with doxycycline., Conclusions: The early administration and proper selection of antibiotics is important for the suppression of inflammatory cytokine production. These findings indicate that antibiotic therapy will not only work in treating infections but might also be useful in treating reactive arthritis secondary to the infection., (Copyright 2007 S. Karger AG, Basel.)

The synthesis of meso-diaminopimelic acid (m-DAP) in bacteria is essential for both peptidoglycan and lysine biosynthesis. From genome sequencing data, it was unclear how bacteria of the Chlamydiales order would synthesize m-DAP in the absence of dapD, dapC, and dapE, which are missing from the genome. Here, we assessed the biochemical capacity of Chlamydia trachomatis serovar L2 to synthesize m-DAP. Expression of the chlamydial asd, dapB, and dapF genes in the respective Escherichia coli m-DAP auxotrophic mutants restored the mutants to DAP prototrophy. Screening of a C. trachomatis genomic library in an E. coli DeltadapD DAP auxotroph identified ct390 as encoding an enzyme that restored growth to the Escherichia coli mutant. ct390 also was able to complement an E. coli DeltadapD DeltadapE, but not a DeltadapD DeltadapF mutant, providing genetic evidence that it encodes an aminotransferase that may directly convert tetrahydrodipicolinate to L,L-diaminopimelic acid. This hypothesis was supported by in vitro kinetic analysis of the CT390 protein and the fact that similar properties were demonstrated for the Protochlamydia amoebophila homologue, PC0685. In vivo, the C. trachomatis m-DAP synthesis genes are expressed as early as 8 h after infection. An aminotransferase activity analogous to CT390 recently has been characterized in plants and cyanobacteria. This previously undescribed pathway for m-DAP synthesis supports an evolutionary relationship among the chlamydiae, cyanobacteria, and plants and strengthens the argument that chlamydiae synthesize a cell wall despite the inability of efforts to date to detect peptidoglycan in these organisms.

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3beta to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

Chlamydiae are obligate intracellular bacterial pathogens that replicate solely within the confines of a membrane-bound vacuole termed an inclusion. Within this protected organelle, chlamydiae acquire host-cell-derived biosynthetic precursors necessary for intracellular subsistence, yet the mechanisms and pathways responsible for this acquisition remain elusive. The present study identifies an interaction between the chlamydial inclusion and multivesicular bodies, complex organelles pivotal in protein and lipid transport that are positioned along the endosome-lysosome pathway, and intersect the exocytic pathway in various cell types. Resident protein and lipid constituents of multivesicular bodies colocalized with intracellular chlamydiae, with direct delivery of the resident protein CD63 to the chlamydial inclusion. Interruption of trafficking from multivesicular bodies by pharmacological inhibitors and exogenous antibodies subsequently disrupted sphingolipid delivery to the maturing chlamydial inclusion and intracellular bacterial growth. This study identifies a trafficking pathway from CD63-positive multivesicular bodies to the bacterial inclusion, a novel interaction that provides essential lipids necessary for maintenance of a productive intracellular infection.

Background: Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes., Results: C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) beta-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands., Conclusions: C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with beta-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.

During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia-associated arthritis. Hep-2 cells were infected with K serovar C. trachomatis and harvested at t = 0-48 h post-infection (p.i; active). Human monocytes were infected similarly and harvested at t = 1-7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep-2 cells from 11 to 48 h p.i; ftsK and ftsW, related to cell division, were expressed similarly. Real-time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep-2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1-7 p.i. and were weakly expressed in patient samples. Real-time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.

The number of inclusion-forming units (IFUs) observed in quantitative chlamydial cultures may be a surrogate for infectivity or transmissibility. Therefore, we conducted a cross-sectional study of 11,034 patients with Chlamydia trachomatis infection who presented to the Seattle-King County public health department clinics between 1988 and 1996, to determine relationships between the number of IFUs observed in culture and sex, age, race, and serovar class. Of the 11,034 cases of infection we studied, 6801 (62%) were cervical infections in women, and 4233 (38%) were urethral infections in men. The median count was 450 IFU for women and 72 IFU for men (P<.001). Overall, both men and women infected with B-class serovars had significantly higher IFU counts than did those infected with C-class serovars (P<.001). The median IFU count fell consistently with increasing age for both women (625 IFU for those <16 years old to 185 IFU for those >30 years old; P<.001) and men (210 IFU for those <16 years old to 40.5 IFU for those >30 years old; P<.001). We found, by use of multiple regression analysis, that sex, age, race, and serovar class remained independently related to IFU count, with counts being highest among young white women infected with B-class serovars.

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium with a unique developmental cycle. Within the host cell cytoplasm, it resides within a membrane-bound compartment, the inclusion. A distinguishing characteristic of the C. trachomatis life cycle is the fusion of the chlamydia-containing inclusions with each other in the host cell cytoplasm. We report that fusion of inclusions does not occur at 32 degreesC in multiple mammalian cell lines and with three different serovars of C. trachomatis. The inhibition of fusion was inclusion specific; the fusion with sphingolipid-containing secretory vesicles and the interaction with early endosomes were unaffected by incubation at 32 degreesC. The inhibition of fusion of the inclusions was not primarily the result of delayed maturation of the inclusion, as infectious progeny was produced in host cells incubated at 32 degreesC, and the unfused inclusions remained competent to fuse up to 48 h postinfection. The ability to reverse the inhibition of fusion by shifting the infected cells from 32 to 37 degreesC allowed the measurement of the rate and the time of fusion of the inclusions after entry of the bacteria. Most significantly, we demonstrate that fusion of inclusions with each other requires bacterial protein synthesis and that the required bacterial protein(s) is present, but inactive or not secreted, at 32 degreesC.

One hundred female patients clinically diagnosed as pelvic inflammatory disease (PID) were studied for the presence of chlamydial infection by cytology and antigen detection. Cervical smears stained by Giemsa revealed inclusion bodies, only in 3 percent of cases. While using Immunocomb Enzyme Immunoassay (EIA) test, antigen was detected in 13% of cases, thereby showing that antigen detection is a better method than cell cytology. A significant correlation with the low socioeconomic status and younger age group was seen in patients showing presence of Chlamydia trachomatis antigen.

Chlamydia trachomatis, an obligate intracellular parasite and a major human pathogen, invades eukaryotic host cells and replicates within a membrane-bound compartment (termed the vacuole or inclusion) in the cytoplasm of the host cell. In this report, we describe in detail the characteristics of the vacuole throughout the chlamydial life cycle in terms of the endocytic pathway, as determined by epifluorescent and confocal immunofluorescence microscopy. By indirect immunofluorescence, the transferrin receptor (TfR), a component of early endosomes, and the cation-independent mannose-6-phosphate receptor (CI-M6PR), a component of late endosomes, were found in close association with the chlamydial vacuole as early as 4 h postinfection (hpi) and as late as 20 hpi. Fluorescein isothiocyanate (FITC)-labeled Tf was also found to colocalize with the vacuole at 4, 12, and 20 hpi, indicating that exogenously added ligands can be transported to the region of the vacuole. Antibodies to several different lysosomal proteins failed to label the chlamydial vacuole at any time point during the life cycle. Indirect immunofluorescence of cells infected with chlamydiae stained with an antibody to the trans-Golgi network (TGN) protein TGN38 demonstrated that in infected cells, the integrity and structure of the TGN was altered. The rates of Tf recycling in infected and uninfected cells were compared by fluorescence microscopy and quantitated with 125I-Tf. While the rate of FITC-Tf recycling from endocytic compartments in chlamydia-infected cells did not appear different from that of uninfected cells, a small pool of FITC-Tf that had accumulated adjacent to the chlamydial vacuole recycled at a slower rate. Quantitation of Tf recycling with 125I-Tf showed that Tf was recycled more slowly in infected cells than in uninfected cells. The altered distribution of several endocytic pathway markers and the slowed Tf recycling are consistent with the hypothesis that the chlamydial vacuole interacts with the endocytic pathway of the host. These results furthermore suggest that the chlamydial vacuole does not correspond to a canonical endocytic compartment but that it is a unique and dynamic organelle that shares several characteristics with recycling endosomes of the host cell. Interactions with the early and/or late endosomal compartments, in addition to the Golgi apparatus, may provide a source of membrane or nutrients for the replicating organisms.

The prevalence of oral contraceptive use in association with chlamydial pelvic inflammatory disease (PID) and the presence of anti-chlamydial IgG and IgA in a population of 144 hospitalized and outpatient subjects with a standard diagnosis of PID was studied. The rates of chlamydial PID and IgA detection were 15.3% (22/144) and 13.9% (20/144), respectively. After stratification for age, number of pregnancies, and lifetime sexual partners, the rates of chlamydial PID (odds ratio = 0.30, 95% CI = 0.10 - 0.89) and IgA detection (odds ratio = 0.23, 95% CI = 0.07 - 0.73) were lower among previous or current oral contraceptive users than in women who had never used birth control methods. Analyses of linear trend indicated a negative association between increasing duration of exposure to hormonal contraception and anti-chlamydial IgG and IgA. This study confirms that among patients with chlamydial PID, the frequency of oral contraceptive use is lower than that in patients with PID of other etiology. Serologic studies suggest a possible relationship between hormonal contraception and changes in immune response or susceptibility to chlamydial infection.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.

The aim of this work is to realise an ultra structural study of the action of antibiotics (doxycycline, erythromycin and ofloxacin) on the intracellular growth cycle of chlamydia. All assays were done on McCoy cells. Antibiotics were added to the culture medium two hours after inoculation with C. trachomatis. 24 and 48 hours, cells were removed and treated by standard procedure for transmission electron microscopy. No difference between control-cells and cells treated with doxycycline, erythromycin (0.015 mg/l) and ofloxacin (0.15 mg/l), was observed in the inclusions (number and size). However 48 hours, the quantity of elementary bodies (E.B.) was lower in treated culture. The organisms were not morphologically different from those seen in untreated culture. At higher concentrations of doxycycline and erythromycin (0.03 mg/l - 0.06 mg/l), the inclusions were smaller. Reticulate bodies (RB) were each in separate inclusion indicating that no fission has taken place. At 48 hours, no infectious forms (EB) were observed. Small or large RBs appeared with an apparent normal cytoplasmic membrane within an inflated cell wall. With ofloxacin (0.3 - 0.6 mg/l), inclusions contained enlarged and sometimes empty bacterial cells. Doxycycline and erythromycin (0.03 mg/l) inhibit the division of RBs and there maturation to EBs Ofloxacin (0.3 mg/l) induces the production of large abnormal forms which may not be able to revert towards viable bacteria.

The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.

Previous studies comparing the cytology brush (Cytobrush) with swabs for the detection of Chlamydia trachomatis from the uterine cervix have yielded divided results. The authors carried out a retrospective analysis at the University of South Florida Student Health Service to evaluate the effect of a change in specimen collection technique on C trachomatis prevalence estimates in female university students. Samples were collected from 1,003 women in 1989, using a small Dacron swab, and 1,229 women in 1990, using a brush; the samples were tested by means of the Pathfinder Enzyme Immunoassay (EIA) detection kit. Positive test results were 5.88% for 1989 and 11.66% for 1990. Actual C trachomatis prevalence for 1989 and 1990 was stable, as determined by three control groups. The results suggest that the cytology brush is more effective than the Dacron swab for the detection of C trachomatis in the uterine cervix when it is used with the Pathfinder EIA test kit.

This investigation compared a rapid enzyme immunoassay test and an enzyme-amplified immunoassay test with culture for the noninvasive detection of Chlamydia trachomatis urethritis in men. Urine specimens from 108 male subjects were evaluated for the presence of C trachomatis antigen, using the Kodak Surecell Chlamydia rapid enzyme immunoassay test and IDEIA Chlamydia enzyme-amplified immunoassay test. The test results were then compared with urethral C trachomatis culture and urethral Gram's stain analyses. Performance characteristics of the two tests were similar. The rapid chlamydial enzyme immunoassay test demonstrated several clinical advantages. Appropriate use of these tests may decrease patient morbidity and clarify organism etiology for enhanced specific medical care of urethritis in men.

From highly purified elementary bodies (EBs) of Chlamydia trachomatis, we have identified a protein of 38 kDa that selectively binds to monolayer cultures of HeLa cells. This protein, which we have named the chlamydial cytadhesin (CCA), is present on the surface of the EBs of three C. trachomatis serovars (B, E, and L1) that were examined. Localization of the CCA at the surface was confirmed by its ability to be labeled when viable EBs were iodinated and by its absence in preparations from trypsin-treated EBs. Viable EBs, but not heated or trypsin-treated EBs, inhibited the binding of the CCA to HeLa cells, indicating competition for a common receptor on the host cell membrane. A dose-dependent inhibition of adherence of radioactive EBs to HeLa cells was effected by extracts containing the CCA. This inhibition occurred even with extracts prepared from the EB of heterologous serovars. However, no inhibition could be demonstrated with extracts prepared from heat-treated EBs. Heat treatment of the extract resulted in the loss of ability of the CCA to bind to the host cells. HeLa cells preincubated with CCA-containing chlamydial extract showed reduced ability to bind labeled EBs and to develop cytoplasmic inclusions after infection. This protective activity was lost after exposure of the extract to heat. These findings indicate that the CCA is a thermolabile surface-exposed chlamydial adhesin; it may be useful in the development of vaccines for diseases caused by the pathogenic bacterium.

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported.

The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells.

Indirect immunofluorescence (IF) staining, using a monoclonal antibody, detected two- to fourfold more inclusions than did iodine staining. Of 274 clinical specimens, 53 (19.3%) were positive by IF on passage 1 as compared with 33 (12%) by iodine staining (P less than 0.005). IF-stained inclusions in McCoy cells in the bottom of microculture wells were readily viewed with a long-focal-length objective at a magnification of 250 X.

The cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1, 2d]-thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1, 2d]thiazolium bromide, interacts with several classes of anionic polymers, exhibiting metachromasia. We were able to stain various kinds of microorganisms with this dye. Gram-negative bacteria were stained reddish purple, while Gram-positive bacteria were stained violet or bluish purple. Stains of molds were of various colors. Yeast vegetative cells were stained reddish purple, but zygotic asci were bluish. Chlamydia trachomatis inclusions, which are surrounded by cytoplasmic membranes, were also stained red. Microorganism and cell stains have different features and can be identified also by use of fluorescent microscopy. The new staining method we report here is rapid and simple enough for routine microscopical examinations of smears of clinical specimens including microorganisms.

McCoy cells treated with cycloheximide, iododeoxyuridine, and DEAE-dextran and untreated McCoy cells were inoculated with two stock strains of Chlamydia trachomatis and with 231 urethral specimens from men, 53 (23%) of which contained C. trachomatis. Isolation rates, number and quality of inclusions, and quality of the cell monolayers were compared. There were no significant differences between the isolation rates in the four systems, although the most isolations were made in the untreated and cycloheximide-treated cells. Cycloheximide-treated cells produced, from both the clinical specimens and the two stock strains, significantly more inclusions than any of the other systems. The monolayer of the cycloheximide-treated cells and the inclusions that grew in these cells were optimal for examination and detection of C. trachomatis.

Chlamydiae are obligately intracellular procaryotic parasites, and their activities as agents of human disease are determined to a large degree by their intracellular way of life. The inside of a host cell is a hostile environment, and few microorganisms survive and multiply intracellularly. Those that do have evolved adaptations that fit them for life inside other cells. Apart from the viruses, chlamydiae are the infectious agents most highly adapted to intracellular life. Of all the properties of chlamydiae, the ones most likely to determine their pathogenic potential are those that reflect their adaptations to life inside host cells. Wherever possible, these chlamydial activities will be indentified and described.