Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase

In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ∼2.5-MDa (1) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2, 3). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4, 5). The 19 S regulatory particle consists of at least 17 different subunits (1, 6). Nine of these subunits form a “lid,” whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7–10) as well as cross-linking experiments (11, 12) have demonstrated that the six homologous ATPases are associated with the α rings of the 20 S particle.Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15). In both PAN and the 19 S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the α ring through which substrates enter the 20 S particle (15–17). Because this portal is quite narrow (18–20), only extended polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded by the associated ATPase complex to be translocated and digested within the 20 S particle.PAN and the six ATPases found at the base of the 19 S particle are members of the AAA+ superfamily of multimeric ATPases which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (8, 21). For mechanistic studies of the roles of ATP, the simpler archaeal PAN-20 S system offers many technical advantages over the much more complex 26 S proteasome. For example, prior studies of PAN (17, 22) demonstrated that unfolding of globular substrates (e.g. green fluorescent protein-ssrA) requires ATP hydrolysis. The same was also shown for the Escherichia coli ATP-dependent proteases ClpXP (23) and ClpAP (24). We have also shown that unfolding by PAN can take place on the surface of the ATPase ring in the absence of translocation (15). Thus, unfolding seems to proceed independently from protein translocation into the 20 S proteolytic particle. It is noteworthy that other studies suggest that proteins are unfolded by energy-dependent translocation through the ATPase ring (25, 26). These studies have suggested that the translocation of an unfolded polypeptide from the ATPase into the 20 S core is an active process that is coupled to ATP hydrolysis. A key to underline a detailed molecular mechanism for substrate binding, unfolding, and translocation by the proteasome regulatory ATPase complex is improved understanding of its architecture and the nucleotide-dependent structural transitions that afford these functions.To date we and others have failed to generate micrographs suitable for three-dimensional reconstruction of PAN using single-particle EM analysis. Likewise, structural information regarding the three-dimensional architecture and subunit organization within the 19 S particle is very limited. In fact, high resolution three-dimensional information on the 19 S complex is not yet available. Most knowledge available is based on cross-linking experiments (11, 12) as well as EM structural analysis (7–10), which provided a three-dimensional model outline of the general architecture of the 26 S complex. Unlike the 19 S complex, the structure of the 20 S subcomplex was determined by x-ray crystallography (18, 19). In contrast to the highly homogenous structure of the 20 S complex, the structural heterogeneity and flexibility of the 19 S subcomplex is presumably reflected in multiple conformations, which in turn also contribute to the difficulty in generating a high resolution three-dimensional structural model of the 26 S proteasome. Accordingly, the initial goal of this study was to generate a three-dimensional model of PAN that will allow us to determine its general architecture and to correlate unique conformational transitions within this ATPase with the nucleotide state of the complex (i.e. in the presence of ATPγS, ADP, or in the absence of nucleotides).Smith et al. (27) suggested a general architecture for the PAN-20 S complex based on two-dimensional averaging of a Thermoplasma acidophilum (TA)³ 20 S proteasome and Methanococcus jannaschii (MJ) PAN hybrid complex in the presence of ATPγS. Based on side-view projections of that complex, these authors proposed that PAN assumes an overall structure similar to E. coli HslU (28–30).We realized that although PAN appears heterogeneous in electron micrographs, it does not occupy all possible orientations when adsorbed to carbon-coated electron microscopy (EM) grids, a prerequisite for single particle analysis. This problem was overcome by applying electron tomography in conjunction with a three-dimensional averaging procedure that accounts for the missing wedge in the Fourier space of electron tomograms (31, 32). The three-dimensional model generated revealed an unexpected architecture leading to a possible molecular mechanism describing the function of PAN and presumably the 19 S ATPases.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry

Ribosomal protein S1 has been shown to be a significant effector of prokaryotic translation. The protein is in fact capable of efficiently initiating translation, regardless of the presence of a Shine-Dalgarno sequence in mRNA. Structural insights into this process have remained elusive, as S1 is recalcitrant to traditional techniques of structural analysis, such as x-ray crystallography. Through the application of protein cross-linking and high resolution mass spectrometry, we have detailed the ribosomal binding site of S1 and have observed evidence of its dynamics. Our results support a previous hypothesis that S1 acts as the mRNA catching arm of the prokaryotic ribosome. We also demonstrate that in solution the major domains of the 30S subunit are remarkably flexible, capable of moving 30–50Å with respect to one another.Initiation of translation is often the rate-limiting step of protein biosynthesis (1). In prokaryotes, this process is widely recognized to be directed by the Shine-Dalgarno (S.D.)¹ sequence of mRNA and its complementation with the 3′ end of 16S rRNA (2). However, binding of the S.D. sequence to the ribosome is not obligatory for initiation. Ribosomal protein S1, widely conserved in prokaryotes, (3) has been shown to efficiently initiate translation, regardless of the presence of an S.D. sequence (4, 5).S1 is a strikingly atyptical ribosomal protein, being both the largest (61 kDa) and the most acidic (pI 4.7) (6). The protein is composed of six homologous repeats each forming beta barrel domains (3) that in solution comprise a highly elongated structure spanning up to ca. 230 Å (7). This length is comparable to the diameter of the ribosome itself. In addition to these anomalous characteristics, S1 is also one of only two ribosomal proteins that has been attributed functional significance (6). Ribosomal protein S1, for instance, has no apparent role in the assembly of the ribosome, (2) yet is critical for translation in E. coli (8, 9). The functional significance of S1 is related to its most pronounced characteristic, the ability to simultaneously bind mRNA and the ribosome. Analysis of fragments produced by limited proteolysis and chemical cleavage of S1 has shown that an N-terminal fragment of S1 (residues 1–193) binds the ribosome (10) but not RNA (11). Likewise, a C-terminal fragment (res 172–557) binds RNA (12, 13) but not the ribosome (6, 10). By nature of this bi-functional structure, S1 enhances the E. coli ribosome''s affinity for RNA ∼5000 fold (14) and can directly mediate initiation of translation by binding the 5′ UTR of mRNA (4, 5). These observations have led to the hypothesis that S1 acts as a catching arm for the prokaryotic ribosome, working to bring mRNA to the proximity of the ribosome and thereby facilitate initiation (6).Unfortunately, structural analyses capturing how S1 is able to function in this manner remain elusive. A high-resolution crystal structure of ribosome bound S1, or even free S1, does not exist, because S1 is recalcitrant to crystallography (6). Preparation of ribosomes for x-ray crystallography actually involves the deliberate removal of ribosomal protein S1 as a means to improve the reproducibility of crystallization and the quality of the ribosome crystals formed (15–17). The structure and interactions of the protein have nevertheless intrigued structural biologists for decades. However, studies completed to date have failed to convincingly demonstrate the interaction between S1 and the rest of the 30S subunit, because they were incapable of localizing the individual S1 domains (16, 18–20).We have studied the binding of S1 to the 30S subunit by combining cross-linking with mass spectrometry. Chemical cross-linking has long been appreciated as a technique to probe protein-protein interactions (21, 22). With the advent of modern mass spectrometers, it can be very effectively employed to confidently identify the exact residues involved in linkages (23–28). In most cross-linking analyses, protein residues are targeted for covalent modification with a molecule that contains two reactive groups separated by a spacer arm of known length. Only protein residues closer than the length of the spacer arm are capable of being linked. Identification of cross-linked residues thereby provides distance constraints for structural modeling. In this work, the novel amidinating protein cross-linker, DEST (diethyl suberthioimidate), was employed (29, 30). This amine reactive reagent, unlike commercially available reagents, preserves the native basicity of the residues it modifies while being effective at physiological pH. Use of the reagent is unlikely to perturb protein structure and the modifications it imparts are compatible with ionization for mass spectrometry. We have additionally shown that the cross-links it forms can be efficiently enriched from other components of proteolytic digests using strong cation exchange (SCX) chromatography, (30) and that DEST cross-linking of ribosomes yields structural information in excellent agreement with x-ray crystallography (29). Although DEST is an 11Å spacer arm cross-linker, it links alpha carbons up to 24Å apart because of the length and flexibility of lysine side chains. Nevertheless, this is sufficient resolution to approximate the binding positions of the 10kDa domains of S1. Furthermore, multiple cross-linking of a single domain significantly enhances the resolution with which it can be localized.Here, through the application of protein cross-linking and high resolution mass spectrometry, we show that S1 binds to the 30S subunit near the anti-S.D. motif of the 16S rRNA, demonstrate that it is highly elongated even when bound to the ribosome, and provide evidence that its C-terminal mRNA binding region is remarkably dynamic. Our results thus indicate S1 is structurally poised, as previously hypothesized, (6) to act as the mRNA catching arm of the prokaryotic ribosome.     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.