Chlamydia trachomatis induces disassembly of the primary cilium to promote the intracellular infection.

Chlamydia trachomatis is a clinically important bacterium that infects epithelial cells of the genitourinary and respiratory tracts and the eye. These differentiated cells are in a quiescent growth state and have a surface organelle called a primary cilium, but the standard Chlamydia cell culture infection model uses cycling cells that lack primary cilia. To investigate if these differences are relevant, we performed infections with host cells that have a primary cilium. We found that C. trachomatis caused progressive loss of the primary cilium that was prevented by disrupting Aurora A (AurA), HDAC6 or calmodulin, which are components of the cellular cilia disassembly pathway. Stabilization of the primary cilium by targeting this pathway caused a large reduction in infectious progeny although there were no changes in chlamydial inclusion growth, chlamydial replication or the ultrastructural appearance of dividing and infectious forms (RBs and EBs, respectively). Thus, the presence of a primary cilium interfered with the production of infectious EBs at a late step in the developmental cycle. C. trachomatis infection also induced quiescent cells to re-enter the cell cycle, as detected by EdU incorporation in S-phase, and Chlamydia-induced cilia disassembly was necessary for cell cycle re-entry. This study therefore describes a novel host-pathogen interaction in which the primary cilium limits a productive Chlamydia infection, and the bacterium counteracts this host cell defense by activating the cellular cilia disassembly pathway.

Differential Effects of Small Molecule Inhibitors on the Intracellular Chlamydia Infection.

Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis. We compared published and novel inhibitors and showed that they can differentially alter inclusion size, chlamydial number and infectious EB production, and that these effects can vary over the course of the intracellular infection. Our results provide the justification for analysis with multiple assays performed either at the end of the infection or over a time course. We also show that this approach has the potential to identify the particular step in the developmental cycle that is impacted by the inhibitor. We furthermore propose that the magnitude of inhibitor-induced progeny defects are best quantified and compared by using a new value called maximal progeny production (Progenymax). As a demonstration of the validity of this systematic approach, we applied it to inhibitors of Akt and AMPK, which are host kinases involved in lipid synthesis and cholesterol trafficking pathways. Both inhibitors reduced EB production, but Akt disruption primarily decreased RB-to-EB conversion while AMPK inhibition paradoxically enhanced RB replication. IMPORTANCE Chlamydia is the most reported cause of bacterial, sexually transmitted infection in the United States. This bacterium infects human cells and reproduces within a cytoplasmic inclusion via an unusual developmental cycle involving two specialized chlamydial forms. Small molecule compounds have been reported to negatively affect the inclusion as well as chlamydial replication and infectious progeny production, but we showed that these effects can be discordant and vary over the course of the 48- to 72-hour long intracellular infection. We propose approaches to analyze these nonuniform effects, including measurements at the end of the intracellular infection, and more detailed analysis with multiple assays performed over the course of the developmental cycle. We then applied this approach to investigate and compare the anti-chlamydial effects of two inhibitors that alter host lipid synthesis and cholesterol trafficking.

A Reverse Genetic Approach for Studying sRNAs in Chlamydia trachomatis.

sRNAs are noncoding transcripts that play critical roles in posttranscriptional regulation in prokaryotes. In the intracellular bacterium Chlamydia, sRNAs have been identified, but functional studies have been limited to an E. coli heterologous system. We have developed an inducible sRNA overexpression system in Chlamydia trachomatis and used it to screen putative sRNAs for effects on the Chlamydia developmental cycle, which involves conversion between replicating (RB) and infectious (EB) chlamydial forms. Overexpression of 4 of 13 C. trachomatis sRNAs decreased production of infectious EBs. We performed detailed characterization of CtrR3 and CtrR7, the two sRNAs that caused the largest progeny defects in our screen. By quantifying chlamydial number and infectious progeny, and by visualizing chlamydial forms using electron microscopy, we showed that overexpression of CtrR3 prevented RB-to-EB conversion, whereas CtrR7 overexpression blocked bacterial replication. We also describe a workflow that allowed us to identify the mRNA targets of CtrR3 in Chlamydia. We first used MS2 aptamer affinity purification coupled with RNA sequencing as an unbiased approach to isolate interacting mRNAs. We then prioritized candidates based on sequence complementarity to the CtrR3 target recognition sequence, which we had identified with bioinformatic and mutational analyses. Finally, we tested putative targets with translational fusion assays in E. coli and C. trachomatis. Using this integrated approach, we provide experimental evidence that YtgB and CTL0389 are mRNA targets of CtrR3 in Chlamydia. These findings demonstrate how our C. trachomatis sRNA overexpression system can be used to investigate the functions and mRNA targets of chlamydial sRNAs. IMPORTANCE Small RNAs (sRNAs) are a class of regulatory RNAs that play important roles in bacterial physiology and pathogenesis. In the intracellular bacterium Chlamydia, however, sRNAs are poorly understood, and functional studies have been limited to a heterologous system. In this study, we developed a genetic system for studying sRNAs in Chlamydia trachomatis and used it to identify four chlamydial sRNAs whose overexpression decreased the production of infectious bacteria. We also successfully utilized this genetic system to determine the target recognition sequence and mRNA targets of an uncharacterized, chlamydial sRNA named CtrR3. Overall, this work offers a generalizable approach for investigating the role of chlamydial sRNAs in their native organism.

Chlamydia and HPV induce centrosome amplification in the host cell through additive mechanisms.

Based on epidemiology studies, Chlamydia trachomatis has been proposed as a co-factor for human papillomavirus (HPV) in the development of cervical cancer. These two intracellular pathogens have been independently reported to induce the production of extra centrosomes, or centrosome amplification, which is a hallmark of cancer cells. We developed a cell culture model to systematically measure the individual and combined effects of Chlamydia and HPV on the centrosome in the same host cell. We found that C. trachomatis caused centrosome amplification in a greater proportion of cells than HPV and that the effects of the two pathogens on the centrosome were additive. Furthermore, centrosome amplification induced by Chlamydia, but not by HPV, strongly correlated with multinucleation and required progression through mitosis. Our results suggest that C. trachomatis and HPV induce centrosome amplification through different mechanisms, with the chlamydial effect being largely due to a failure in cytokinesis that also results in multinucleation. Our findings provide support for C. trachomatis as a co-factor for HPV in carcinogenesis and offer mechanistic insights into how two infectious agents may cooperate to promote cancer. TAKE AWAYS: • Chlamydia and HPV induce centrosome amplification in an additive manner. • Chlamydia-induced centrosome amplification is linked to host cell multinucleation. • Chlamydia-induced centrosome amplification requires cell cycle progression. • Chlamydia and HPV cause centrosome amplification through different mechanisms. • This study supports Chlamydia as a co-factor for HPV in carcinogenesis.

The Small Molecule H89 Inhibits Chlamydia Inclusion Growth and Production of Infectious Progeny.

Chlamydia is an obligate intracellular bacterium and the most common reportable cause of human infection in the United States. This pathogen proliferates inside a eukaryotic host cell, where it resides within a membrane-bound compartment called the chlamydial inclusion. It has an unusual developmental cycle, marked by conversion between a replicating form, the reticulate body (RB), and an infectious form, the elementary body (EB). We found that the small molecule H89 slowed inclusion growth and decreased overall RB replication by 2-fold but caused a 25-fold reduction in infectious EBs. This disproportionate effect on EB production was mainly due to a defect in RB-to-EB conversion and not to the induction of chlamydial persistence, which is an altered growth state. Although H89 is a known inhibitor of specific protein kinases and vesicular transport to and from the Golgi apparatus, it did not cause these anti-chlamydial effects by blocking protein kinase A or C or by inhibiting protein or lipid transport. Thus, H89 is a novel anti-chlamydial compound that has a unique combination of effects on an intracellular Chlamydia infection.

Stochastic Chlamydia Dynamics and Optimal Spread.

Chlamydia trachomatis is an important bacterial pathogen that has an unusual developmental switch from a dividing form (reticulate body or RB) to an infectious form (elementary body or EB). RBs replicate by binary fission within an infected host cell, but there is a delay before RBs convert into EBs for spread to a new host cell. We developed stochastic optimal control models of the Chlamydia developmental cycle to examine factors that control the number of EBs produced. These factors included the probability and timing of conversion, and the duration of the developmental cycle before the host cell lyses. Our mathematical analysis shows that the observed delay in RB-to-EB conversion is important for maximizing EB production by the end of the intracellular infection.

Function of Golgi-centrosome proximity in RPE-1 cells.

The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.

Replication-dependent size reduction precedes differentiation in Chlamydia trachomatis.

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.

Spatial analysis of Cdc42 activity reveals a role for plasma membrane-associated Cdc42 in centrosome regulation.

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni-toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase.

The daughter centriole controls ciliogenesis by regulating Neurl-4 localization at the centrosome.

The two centrioles of the centrosome differ in age and function. Although the mother centriole mediates most centrosome-dependent processes, the role of the daughter remains poorly understood. A recent study has implicated the daughter centriole in centriole amplification in multiciliated cells, but its contribution to primary ciliogenesis is unclear. We found that manipulations that prevent daughter centriole formation or induce its separation from the mother abolish ciliogenesis. This defect was caused by stabilization of the negative ciliogenesis regulator CP110 and was corrected by CP110 depletion. CP110 dysregulation may be caused by effects on Neurl-4, a daughter centriole-associated ubiquitin ligase cofactor, which was required for ciliogenesis. Centrosome-targeted Neurl-4 was sufficient to restore ciliogenesis in cells with manipulated daughter centrioles. Interestingly, early during ciliogenesis, Neurl-4 transiently associated with the mother centriole in a process that required mother-daughter centriole proximity. Our data support a model in which the daughter centriole promotes ciliogenesis through Neurl-4-dependent regulation of CP110 levels at the mother centriole.

Induction and inhibition of CPAF activity during analysis of Chlamydia-infected cells.

Studies of the chlamydial protease CPAF have been complicated by difficulties in distinguishing bona fide intracellular proteolysis from in vitro proteolysis. This confounding issue has been attributed to CPAF activity in lysates from Chlamydia-infected cells. We compared three methods that have been used to inhibit in vitro CPAF-mediated proteolysis: (1) pre-treatment of infected cells with the inhibitor clasto-lactacystin, (2) direct cell lysis in 8 M urea and (3) direct lysis in hot 1% SDS buffer. We identified a number of experimental conditions that reduce the effectiveness of each method in preventing CPAF activity during lysate preparation. The amount of in vitro proteolysis in a lysate was variable and depended on factors such as the specific substrate and the time in the intracellular infection. Additionally, we demonstrated for the first time that artifactual CPAF activity is induced before cell lysis by standard cell detachment methods, including trypsinization. Protein analysis of Chlamydia-infected cells therefore requires precautions to inhibit CPAF activity during both cell detachment and lysate preparation, followed by verification that the cell lysates do not contain residual CPAF activity. These concerns about artifactual proteolysis extend beyond studies of CPAF function because they have the potential to affect the analyses of host and chlamydial proteins from Chlamydia-infected cells.

Signaling at the Golgi during mitosis.

The Golgi complex of mammalian cells is composed of interconnected stacks of flattened cisternae that form a continuous membrane system in the pericentriolar region of the cell. At the onset of mitosis, this so-called Golgi ribbon is converted into small tubular-vesicular clusters in a tightly regulated fragmentation process, which leads to a temporary loss of the physical Golgi-centrosome proximity. Mitotic Golgi breakdown is required for Golgi partitioning into the two daughter cells, cell cycle progression and may contribute to the dispersal of Golgi-associated signaling molecules. Here, we review our current understanding of the mechanisms that control mitotic Golgi reorganization, its biological significance, and assays that are used to study this process.

Par6γ is at the mother centriole and controls centrosomal protein composition through a Par6α-dependent pathway.

The centrosome contains two centrioles that differ in age, protein composition and function. This non-membrane bound organelle is known to regulate microtubule organization in dividing cells and ciliogenesis in quiescent cells. These specific roles depend on protein appendages at the older, or mother, centriole. In this study, we identified the polarity protein partitioning defective 6 homolog gamma (Par6γ) as a novel component of the mother centriole. This specific localization required the Par6γ C-terminus, but was independent of intact microtubules, the dynein/dynactin complex and the components of the PAR polarity complex. Par6γ depletion resulted in altered centrosomal protein composition, with the loss of a large number of proteins, including Par6α and p150(Glued), from the centrosome. As a consequence, there were defects in ciliogenesis, microtubule organization and centrosome reorientation during migration. Par6γ interacted with Par3 and aPKC, but these proteins were not required for the regulation of centrosomal protein composition. Par6γ also associated with Par6α, which controls protein recruitment to the centrosome through p150(Glued). Our study is the first to identify Par6γ as a component of the mother centriole and to report a role of a mother centriole protein in the regulation of centrosomal protein composition.

CPAF: a Chlamydial protease in search of an authentic substrate.

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.

Divergent synthesis and chemical reactivity of bicyclic lactone fragments of complex rearranged spongian diterpenes.

The synthesis and direct comparison of the chemical reactivity of the two highly oxidized bicyclic lactone fragments found in rearranged spongian diterpenes (8-substituted 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one and 6-substituted 7-acetoxy-2,8-dioxabicyclo[3.3.0]octan-3-one) are reported. Details of the first synthesis of the 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one ring system, including an examination of several possibilities for the key bridging cyclization reaction, are described. In addition, the first synthesis of 7-acetoxy-2,8-dioxabicyclo[3.3.0]octanones containing quaternary carbon substituents at C6 is disclosed. Aspects of the chemical reactivity and Golgi-modifying properties of these bicyclic lactone analogs of rearranged spongian diterpenes are also reported. Under both acidic and basic conditions, 8-substituted 2,7-dioxabicyclo[3.2.1]octanones are converted to 6-substituted-2,8-dioxabicyclo[3.3.0]octanones. Moreover, these dioxabicyclic lactones react with primary amines and lysine side chains of lysozyme to form substituted pyrroles, a conjugation that could be responsible for the unique biological properties of these compounds. These studies demonstrate that acetoxylation adjacent to the lactone carbonyl group, in either the bridged or fused series, is required to produce fragmented Golgi membranes in the pericentriolar region that is characteristic of macfarlandin E.

Par6 alpha interacts with the dynactin subunit p150 Glued and is a critical regulator of centrosomal protein recruitment.

The centrosome contains proteins that control the organization of the microtubule cytoskeleton in interphase and mitosis. Its protein composition is tightly regulated through selective and cell cycle-dependent recruitment, retention, and removal of components. However, the mechanisms underlying protein delivery to the centrosome are not completely understood. We describe a novel function for the polarity protein Par6α in protein transport to the centrosome. We detected Par6α at the centrosome and centriolar satellites where it interacted with the centriolar satellite protein PCM-1 and the dynactin subunit p150(Glued). Depletion of Par6α caused the mislocalization of p150(Glued) and centrosomal components that are critical for microtubule anchoring at the centrosome. As a consequence, there were severe alterations in the organization of the microtubule cytoskeleton in the absence of Par6α and cell division was blocked. We propose a model in which Par6α controls centrosome organization through its association with the dynactin subunit p150(Glued).

The Golgi and the centrosome: building a functional partnership.

The mammalian Golgi apparatus is characterized by a ribbon-like organization adjacent to the centrosome during interphase and extensive fragmentation and dispersal away from the centrosome during mitosis. It is not clear whether this dynamic association between the Golgi and centrosome is of functional significance. We discuss recent findings indicating that the Golgi-centrosome relationship may be important for directional protein transport and centrosome positioning, which are both required for cell polarization. We also summarize our current knowledge of the link between Golgi organization and cell cycle progression.

Golgi-modifying properties of macfarlandin E and the synthesis and evaluation of its 2,7-dioxabicyclo[3.2.1]octan-3-one core.

Golgi-modifying properties of the spongian diterpene macfarlandin E (MacE) and a synthetic analog, t-Bu-MacE, containing its 2,7-dioxabicyclo[3.2.1]octan-3-one moiety are reported. Natural product screening efforts identified MacE as inducing a novel morphological change in Golgi structure defined by ribbon fragmentation with maintenance of the resulting Golgi fragments in the pericentriolar region. t-Bu-MacE, which possesses the substituted 2,7-dioxabicyclo[3.2.1]octan-3-one but contains a tert-butyl group in place of the hydroazulene subunit of MacE, was prepared by chemical synthesis. Examination of the Golgi-modifying properties of MacE, t-Bu-MacE, and several related structures revealed that the entire oxygen-rich bridged-bicyclic fragment is required for induction of this unique Golgi organization phenotype. Further characterization of MacE-induced Golgi modification showed that protein secretion is inhibited, with no effect on the actin or microtubule cytoskeleton being observed. The conversion of t-Bu-MacE and a structurally related des-acetoxy congener to substituted pyrroles in the presence of primary amines in protic solvent at ambient temperatures suggests that covalent modification might be involved in the Golgi-altering activity of MacE.