Metabolic dormancy in Chlamydia trachomatis treated with different antibiotics.

Diseases caused by Chlamydia spp. are often associated with persistent infections. Chlamydial persistence is commonly associated with a unique non-infectious intracellular developmental form, termed an aberrant form. Although infectious chlamydiae can be cultured consistently in cells stressed to aberrancy, their role in persistence is not clear. Recovery from antibiotic stress was explored as a model to determine how survival of non-aberrant chlamydiae, in the presence of fully inhibitory drug concentrations, may participate in persistence. Assays included incubation in quinolones, tetracyclines, or chloramphenicol for differing lengths of time, followed by an extended recovery period in antibiotic-free media. Culturable elementary bodies were not detected during treatment with each antibiotic, but viable and culturable Chlamydia trachomatis emerged after the drug was removed. Time-lapse imaging of live, antibiotic-treated infected cells identified metabolically dormant developmental forms within cells that emerged to form typical productive inclusions. The effects of the increasing concentration of most tested antibiotics led to predictable inhibitory activity, in which the survival rate decreased with increasing drug concentration. In contrast, in fluoroquinolone-treated cells, there was a paradoxical increase in productive development that was directly correlated with drug concentration and inversely associated with aberrant form production. This model system uncovers a unique chlamydial persistence pathway that does not involve the chlamydial aberrant form. The association between productive latency and metabolic dormancy is consistent with models for many bacterial species and may lead to a different interpretation of mechanisms of chlamydial persistence in patients.IMPORTANCEThe life history of most pathogens within the genus Chlamydia relies on lengthy persistence in the host. The most generally accepted model for Chlamydia spp. persistence involves an unusual developmental stage, termed the aberrant form, which arises during conditions that mimic a stressful host environment. In this work, we provide an alternate model for chlamydial persistence in the face of antibiotic stress. This model may be relevant to antibiotic treatment failures in patients infected with C. trachomatis.

Computational Modeling of the Chlamydial Developmental Cycle Reveals a Potential Role for Asymmetric Division.

Chlamydia trachomatis is an obligate intracellular bacterium that progresses through an essential multicell form developmental cycle. Infection of the host is initiated by the elementary body (EB). Once in the host, the EB cell differentiates into the noninfectious, but replication-competent, reticulate body, or RB. After multiple rounds of replication, RBs undergo secondary differentiation, eventually producing newly infectious EBs. Here, we generated paired cell-type promoter reporter constructs and determined the kinetics of the activities of the euo, hctA, and hctB promoters. The paired constructs revealed that the developmental cycle produces at least three phenotypically distinct cell types, the RB (euoprom+), intermediate body (IB; hctAprom+), and EB (hctBprom+). The kinetic data from the three dual-promoter constructs were used to generate two computational agent-based models to reproduce the chlamydial developmental cycle. Both models simulated EB germination, RB amplification, IB formation, and EB production but differed in the mechanism that generated the IB. The direct conversion and the asymmetric production models predicted different behaviors for the RB population, which were experimentally testable. In agreement with the asymmetric production model, RBs acted as stem cells after the initial amplification stage, producing one IB and self-renewing after every division. We also demonstrated that IBs are a transient cell population, maturing directly into EBs after formation without the need for cell division. The culmination of these results suggests that the developmental cycle can be described by a four-stage model, EB germination, RB amplification/maturation, IB production, and EB formation. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for both ocular and sexually transmitted infections. All Chlamydiae are reliant on a complex developmental cycle, consisting of both infectious and noninfectious cell forms. The EB cell form initiates infection, whereas the RB cell replicates. The infectious cycle requires both cell types, as RB replication increases the cell population while EB formation disseminates the infection to new hosts. The mechanisms of RB-to-EB development are largely unknown. Here, we developed unique dual promoter reporters and used live-cell imaging and confocal microscopy to visualize the cycle at the single-cell and kinetic levels. These data were used to develop and test two agent-based models, simulating either direct conversion of RBs to EBs or production of EBs via asymmetric RB division. Our results suggest that RBs mature into a stem cell-like population producing intermediate cell forms through asymmetric division, followed by maturation of the intermediate cell type into the infectious EB. Ultimately, a more complete mechanistic understanding of the developmental cycle will lead to novel therapeutics targeting cell type development to eliminate chlamydial dissemination.

Expression and structure of the Chlamydia trachomatis DksA ortholog.

Chlamydia trachomatis is a bacterial obligate intracellular parasite and a significant cause of human disease, including sexually transmitted infections and trachoma. The bacterial RNA polymerase-binding protein DksA is a transcription factor integral to the multicomponent bacterial stress response pathway known as the stringent response. The genome of C. trachomatis encodes a DksA ortholog (DksACt) that is maximally expressed at 15-20 h post infection, a time frame correlating with the onset of transition between the replicative reticulate body (RB) and infectious elementary body (EB) forms of the pathogen. Ectopic overexpression of DksACt in C. trachomatis prior to RB-EB transitions during infection of HeLa cells resulted in a 39.3% reduction in overall replication (yield) and a 49.6% reduction in recovered EBs. While the overall domain organization of DksACt is similar to the DksA ortholog of Escherichia coli (DksAEc), DksACt did not functionally complement DksAEc. Transcription of dksACt is regulated by tandem promoters, one of which also controls expression of nrdR, encoding a negative regulator of deoxyribonucleotide biosynthesis. The phenotype resulting from ectopic expression of DksACt and the correlation between dksACt and nrdR expression is consistent with a role for DksACt in the C. trachomatis developmental cycle.

Translational gene expression control in Chlamydia trachomatis.

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

The sRNA Regulated Protein DdbA Is Involved in Development and Maintenance of the Chlamydia trachomatis EB Cell Form.

The chlamydial small non coding RNA, IhtA, regulates the expression of both HctA and DdbA, the uncharacterized product of the C. trachomatis L2 CTL0322 gene. HctA is a small, highly basic, DNA binding protein that is expressed late in development and mediates the condensation of the genome during RB to EB differentiation. DdbA is conserved throughout the chlamydial lineage, and is predicted to express a small, basic, cytoplasmic protein. As it is common for sRNAs to regulate multiple mRNAs within the same physiological pathway, we hypothesize that DdbA, like HctA, is involved in RB to EB differentiation. Here, we show that DdbA is a DNA binding protein, however unlike HctA, DdbA does not contribute to genome condensation but instead likely has nuclease activity. Using a DdbA temperature sensitive mutant, we show that DdbAts creates inclusions indistinguishable from WT L2 in size and that early RB replication is likewise similar at the nonpermissive temperature. However, the number of DdbAts infectious progeny is dramatically lower than WT L2 overall, although production of EBs is initiated at a similar time. The expression of a late gene reporter construct followed live at 40°C indicates that late gene expression is severely compromised in the DdbAts strain. Viability assays, both in host cells and in axenic media indicate that the DdbAts strain is defective in the maintenance of EB infectivity. Additionally, using Whole Genome Sequencing we demonstrate that chromosome condensation is temporally separated from DNA replication during the RB to EB transition. Although DdbA does not appear to be directly involved in this process, our data suggest it is a DNA binding protein that is important in the production and maintenance of infectivity of the EB, perhaps by contributing to the remodeling of the EB chromosome.

Single-Inclusion Kinetics of Chlamydia trachomatis Development.

The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an "intrinsic" cell-autonomous program inherent to each RB and an "extrinsic" environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB.IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium's pathogenesis.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis.

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication. Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Impact of Active Metabolism on Chlamydia trachomatis Elementary Body Transcript Profile and Infectivity.

Bacteria of the genus Chlamydia include the significant human pathogens Chlamydia trachomatis and C. pneumoniae All chlamydiae are obligate intracellular parasites that depend on infection of a host cell and transition through a biphasic developmental cycle. Following host cell invasion by the infectious elementary body (EB), the pathogen transitions to the replicative but noninfectious reticulate body (RB). Differentiation of the RB back to the EB is essential to generate infectious progeny. While the EB form has historically been regarded as metabolically inert, maintenance of infectivity during incubation with specific nutrients has revealed active maintenance of the infectious phenotype. Using transcriptome sequencing, we show that the transcriptome of extracellular EBs incubated under metabolically stimulating conditions does not cluster with germinating EBs but rather with the transcriptome of EBs isolated directly from infected cells. In addition, the transcriptional profile of the extracellular metabolizing EBs more closely resembled that of EB production than germination. Maintenance of infectivity of extracellular EBs was achieved by metabolizing chemically diverse compounds, including glucose 6-phosphate, ATP, and amino acids, all of which can be found in extracellular environments, including mucosal secretions. We further show that the EB cell type actively maintains infectivity in the inclusion after terminal differentiation. Overall, these findings contribute to the emerging understanding that the EB cell form is actively maintained through metabolic processes after terminal differentiation to facilitate prolonged infectivity within the inclusion and under host cell free conditions, for example, following deposition at mucosal surfaces.IMPORTANCE Chlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a wide range of diseases in both animal and human hosts. According to the Centers for Disease Control and Prevention, C. trachomatis is the most frequently reported sexually transmitted infection in the United States, costing the American health care system nearly $2.4 billion annually. Every year, there are over 4 million new cases of Chlamydia infections in the United States and an estimated 100 million cases worldwide. To cause disease, Chlamydia must successfully complete its complex biphasic developmental cycle, alternating between an infectious cell form (EB) specialized for initiating entry into target cells and a replicative form (RB) specialized for creating and maintaining the intracellular replication niche. The EB cell form has historically been considered metabolically quiescent, a passive entity simply waiting for contact with a host cell to initiate the next round of infection. Recent studies and data presented here demonstrate that the EB maintains its infectious phenotype by actively metabolizing a variety of nutrients. Therefore, the EB appears to have an active role in chlamydial biology, possibly within multiple environments, such as mucosal surfaces, fomites, and inside the host cell after formation.

Identification of the base-pairing requirements for repression of hctA translation by the small RNA IhtA leads to the discovery of a new mRNA target in Chlamydia trachomatis.

The non-coding small RNA, IhtA expressed by the obligate intracellular human pathogen Chlamydia trachomatis modulates the translation of HctA, a key protein involved in replicative to infectious cell type differentiation. Using a combination of bioinformatics and mutagenesis we sought to identify the base pairing requirement for functional repression of HctA protein expression, with an eye to applying our findings towards the identification of additional targets. IhtA is predicted to fold into a three stem:loop structure. We found that loop 1 occludes the initiation codon of hctA, while loop 2 and 3 are not required for function. This 7 nucleotide region forms G/C rich interactions surrounding the AUG of hctA. Two additional genes in the chlamydial genome, CTL0322 and CTL0097, contained some elements of the hctA:IhtA recognition sequence. The mRNA of both CTL0322and CTL0097 interacted with IhtA in vitro as measured by biolayer interferometry. However, using a CheZ reporter expression system, IhtA only inhibited the translation of CTL0322. The proposed IhtA recognition site in the CTL0322 message contains significant G/C base pairing on either side of the initiation codon while CTL0097 only contains G/C base pairing 3' to the AUG initiation codon. These data suggest that as the functional interacting region is only 6-7nt in length that full translation repression is dependent on the degree of G/C base pairing. Additionally our results indicate that IhtA may regulate multiple mRNAs involved in the chlamydial infectious cycle.

Microinjection of specific anti-IMPDH2 antibodies induces disassembly of cytoplasmic rods/rings that are primarily stationary and stable structures.

BACKGROUND: Our laboratory previously reported interesting rods 3-10 µm long and rings 2-5 µm diameter (RR) in the cytoplasm of mammalian cells. Experimental evidence show that both inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthetase (CTPS) are components of RR structures. Several cell types, including mouse embryonic stem cells, and cell lines, such as mouse 3 T3 and rat NRK, naturally present RR structures, while other cells can present RR when treated with compounds interfering with GTP/CTP biosynthetic pathways. In this study, we aimed to investigate the dynamic behavior of these RR in live cells. RESULTS: RR were detected in >90% of COS-7 and HeLa cells treated with 1 mM ribavirin or 6-Diazo-5-oxo-L-norleucine (DON) for 24 h, and in 75% of COS-7 cells treated with 1 mM mycophenolic acid (MPA) for the same period of time. Microinjection of affinity-purified anti-IMPDH2 antibodies in live COS-7 cells treated with ribavirin, DON, or MPA showed mature forms of RR presented as stable and stationary structures in 71% of cells. In the remaining 29% of cells, RR acquired erratic movement and progressively disassembled into fragments and disappeared within 10 min. The specific stationary state and antibody-dependent disassembling of RR structures was independently confirmed in COS-7 and HeLa cells transfected with GFP-tagged IMPDH2. CONCLUSIONS: This is the first demonstration of disassembly of RR structures upon microinjection of anti-IMPDH2 antibodies that led to the disappearance of the molecular aggregates. The disassembly of RR after microinjection of anti-IMPDH2 antibody further strengthens the notion that IMPDH2 are major building blocks of RR. Using two independent methods, this study demonstrated that the induced RR are primarily stationary structures in live cells and that IMPDH2 is a key component of RR.

The Chlamydia effector TarP mimics the mammalian leucine-aspartic acid motif of paxillin to subvert the focal adhesion kinase during invasion.

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

Multinucleation during C. trachomatis infections is caused by the contribution of two effector pathways.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen and the second leading cause of sexually transmitted infections in the US. Infections cause significant morbidity and can lead to serious reproductive sequelae, including an epidemiological link to increased rates of reproductive cancers. One of the overt changes that infected cells exhibit is the development of genomic instability leading to multinucleation. Here we demonstrate that the induction of multinucleation is not conserved equally across chlamydial species; C. trachomatis L2 caused high levels of multinucleation, C. muridarum intermediate levels, and C. caviae had very modest effects on multinucleation. Our data show that at least two effector pathways together cause genomic instability during infection leading to multinucleation. We find that the highly conserved chlamydial protease CPAF is a key effector for one of these pathways. CPAF secretion is required for the loss of centrosome duplication regulation as well as inducing early mitotic exit. The second effector pathway involves the induction of centrosome position errors. This function is not conserved in three chlamydial species tested. Together these two pathways contribute to the induction of high levels of genomic instability and multinucleation seen in C. trachomatis infections.

Expression of Porphyromonas gingivalis small RNA in response to hemin availability identified using microarray and RNA-seq analysis.

There is a significant body of work suggesting that sRNA-mediated post-transcriptional regulation is a conserved mechanism among pathogenic bacteria to modulate bacterial virulence and survival. Porphyromonas gingivalis is recognized as an etiological agent of periodontitis and implicated in contributing to the development of multiple inflammatory diseases including cardiovascular disease. Using NimbleGen microarray analysis and a strand-specific method to sequence cDNA libraries of small RNA-enriched P. gingivalis transcripts using Illumina's high-throughput sequencing technology, we identified putative sRNA and generated sRNA expression profiles in response to growth phase, hemin availability after hemin starvation, or both. We identified transcripts that mapped to intergenic sequences as well as antisense transcripts that mapped to open reading frames of the annotated genome. Overall, this approach provided a comprehensive way to survey transcriptional activity to discover functionally linked RNA transcripts, responding to specific environmental cues, that merit further investigation.

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

BACKGROUND: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized. RESULTS: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs. CONCLUSIONS: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Chlamydia induces anchorage independence in 3T3 cells and detrimental cytological defects in an infection model.

Chlamydia are gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI) Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV). We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect.

Translation inhibition of the developmental cycle protein HctA by the small RNA IhtA is conserved across Chlamydia.

The developmental cycle of the obligate intracellular pathogen Chlamydia trachomatis serovar L2 is controlled in part by the small non-coding RNA (sRNA), IhtA. All Chlamydia alternate in a regulated fashion between the infectious elementary body (EB) and the replicative reticulate body (RB) which asynchronously re-differentiates back to the terminal EB form at the end of the cycle. The histone like protein HctA is central to RB:EB differentiation late in the cycle as it binds to and occludes the genome, thereby repressing transcription and translation. The sRNA IhtA is a critical component of this regulatory loop as it represses translation of hctA until late in infection at which point IhtA transcription decreases, allowing HctA expression to occur and RB to EB differentiation to proceed. It has been reported that IhtA is expressed during infection by the human pathogens C. trachomatis serovars L2, D and L2b and C. pneumoniae. We show in this work that IhtA is also expressed by the animal pathogens C. caviae and C. muridarum. Expression of HctA in E. coli is lethal and co-expression of IhtA relieves this phenotype. To determine if regulation of HctA by IhtA is a conserved mechanism across pathogenic chlamydial species, we cloned hctA and ihtA from C. trachomatis serovar D, C. muridarum, C. caviae and C. pneumoniae and assayed for rescue of growth repression in E. coli co-expression studies. In each case, co-expression of ihtA with the cognate hctA resulted in relief of growth repression. In addition, expression of each chlamydial species IhtA rescued the lethal phenotype of C. trachomatis serovar L2 HctA expression. As biolayer interferometry studies indicate that IhtA interacts directly with hctA message for all species tested, we predict that conserved sequences of IhtA are necessary for function and/or binding.

Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron-dependent transcriptional repressor.

The regulation of iron homeostasis is essential for most organisms, because iron is required for a variety of conserved biochemical processes, yet can be toxic at high concentrations. Upon experiencing iron starvation in vitro, the obligate intracellular human pathogen Chlamydia trachomatis exhibits elevated expression of a putative iron-transport system encoded by the ytg operon. The third component of the ytg operon, CT069 (YtgCR), encodes a protein with two distinct domains: a membrane-anchored metal ion permease and a diphtheria toxin repressor (DtxR)-like transcriptional repressor. In this report, we demonstrate that the C-terminal domain of CT069 (YtgR) serves as an iron-dependent autorepressor of the ytg operon. Moreover, the nascent full-length metal permease-transcriptional repressor protein was processed during the course of infection, and heterologously when expressed in Escherichia coli. The products produced by heterologous cleavage in E. coli were functional in the repression of a reporter gene downstream of a putative YtgR operator. We report a bona fide mechanism of iron-dependent regulation of transcription in Chlamydia. Moreover, the unusual membrane permease-DNA-binding polypeptide fusion configuration was found in several bacteria. Therefore, the DNA-binding capability and liberation of the YtgR domain from a membrane-anchored permease in C. trachomatis could represent a previously uncharacterized mechanism for prokaryotic regulation of iron-homeostasis.

Chlamydial infection induces host cytokinesis failure at abscission.

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.

Methods to assess beta cell death mediated by cytotoxic T lymphocytes.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 ((51)Cr) to label target cells. These targets are then exposed to effector cells and the release of (51)Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of (51)Cr. As effector cells, we used an activated autoreactive clonal population of CD8(+) Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with (51)Cr labeled target NIT cells for 16 hours, release of (51)Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO(2;), and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes. Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.