The trans-Golgi SNARE syntaxin 6 is recruited to the chlamydial inclusion membrane.

Chlamydia trachomatis is an obligate intracellular pathogen that replicates within a parasitophorous vacuole termed an inclusion. The chlamydial inclusion is isolated from the endocytic pathway but fusogenic with Golgi-derived exocytic vesicles containing sphingomyelin and cholesterol. Sphingolipids are incorporated into the chlamydial cell wall and are considered essential for chlamydial development and viability. The mechanisms by which chlamydiae obtain eukaryotic lipids are poorly understood but require chlamydial protein synthesis and presumably modification of the inclusion membrane to initiate this interaction. A polarized cell model of chlamydial infection has demonstrated that chlamydiae preferentially intercept basolaterally directed, sphingomyelin-containing exocytic vesicles. Here we examine the localization and potential function of trans-Golgi and/or basolaterally associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in chlamydia-infected cells. The trans-Golgi SNARE protein syntaxin 6 is recruited to the chlamydial inclusion in a manner that requires chlamydial protein synthesis and is conserved among all chlamydial species examined. The localization of syntaxin 6 to the chlamydial inclusion requires a tyrosine motif or plasma membrane retrieval signal (YGRL). Thus in addition to expression of at least two inclusion membrane proteins that contain SNARE-like motifs, chlamydiae also actively recruit eukaryotic SNARE-family proteins.

The chlamydial inclusion preferentially intercepts basolaterally directed sphingomyelin-containing exocytic vacuoles.

Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C(6)-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking.

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases.

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.

Proteome and antigen profiling of Coxiella burnetii developmental forms.

A biphasic developmental cycle whereby highly resistant small-cell variants (SCVs) are generated from large-cell variants (LCVs) is considered fundamental to the virulence of Coxiella burnetii, the causative agent of human Q fever. In this study a proteome analysis of C. burnetii developmental forms was conducted to provide insight into their unique biological and immunological properties. Silver-stained gels of SCV and LCV lysates separated by two-dimensional (2-D) gel electrophoresis resolved over 675 proteins in both developmental forms. Forty-eight proteins were greater than twofold more abundant in LCVs than in SCVs, with six proteins greater than twofold more abundant in SCVs than in LCVs. Four and 15 upregulated proteins of SCVs and LCVs, respectively, were identified by mass spectrometry, and their predicted functional roles are consistent with a metabolically active LCV and a structurally resistant SCV. One-dimensional and 2-D immunoblots of cell form lysates probed with sera from infected/vaccinated guinea pigs and convalescent-phase serum from human patients who had recovered from acute Q fever, respectively, revealed both unique SCV/LCV antigens and common SCV/LCV antigens that were often differentially synthesized. Antigens recognized during human infection were identified by mass spectroscopy and included both previously described immunodominant proteins of C. burnetii and novel immunogenic proteins that may be important in the pathophysiology of clinical Q fever and/or the induction of protective immunity.

Chlamydial TARP is a bacterial nucleator of actin.

Chlamydia trachomatis entry into host cells results from a parasite-directed remodeling of the actin cytoskeleton. A type III secreted effector, TARP (translocated actin recruiting phosphoprotein), has been implicated in the recruitment of actin to the site of internalization. To elucidate the role of TARP in actin recruitment, we identified host cell proteins that associated with recombinant GST-TARP fusions. TARP directly associated with actin, and this interaction promoted actin nucleation as determined by in vitro polymerization assays. Domain analysis of TARP identified an actin-binding domain that bears structural and primary amino acid sequence similarity to WH2 domain family proteins. In addition, a proline-rich domain was found to promote TARP oligomerization and was required for TARP-dependent nucleation of new actin filaments. Our findings reveal a mechanism by which chlamydiae induce localized cytoskeletal changes by the translocated effector TARP during entry into host cells.

Maturation of human neutrophil phagosomes includes incorporation of molecular chaperones and endoplasmic reticulum quality control machinery.

A Human neutrophils are an essential component of the innate immune response. Although significant progress has been made toward understanding mechanisms of phagocytosis and microbicidal activity, a comprehensive analysis of proteins comprising neutrophil phagosomes has not been conducted. To that end, we used subcellular proteomics to identify proteins associated with human neutrophil phagosomes following receptor-mediated phagocytosis. Proteins (n = 411 spots) resolved from neutrophil phagosome fractions were identified by MALDI-TOF MS and/or LC-MS/MS analysis. Those associated with phagocytic vacuoles originated from multiple subcellular compartments, including the cytosol, plasma membrane, specific and azurophilic granules, and cytoskeleton. Unexpectedly several enzymes typically associated with mitochondria were identified in phagosome fractions. Furthermore proteins characteristic of the endoplasmic reticulum, including 11 molecular chaperones, were resolved from phagosome preparations. Confocal microscopy confirmed that proteins representing these major subcellular compartments were enriched on phagosomes of intact neutrophils. Notably calnexin and glucose-regulated protein 78 co-localized with gp91(phox) in human neutrophils and were thus likely delivered to phagosomes by fusion of specific granules. We conclude that neutrophil phagosomes have heretofore unrecognized complexity and function, which includes potential for antigen processing events.

Analysis of putative Chlamydia trachomatis chaperones Scc2 and Scc3 and their use in the identification of type III secretion substrates.

The obligate intracellular pathogen Chlamydia trachomatis expresses a type III secretion system (T3SS) which has the potential to contribute significantly to pathogenesis. Based on a demonstrated role of type III secretion (T3S)-specific chaperones in the secretion of antihost proteins by gram-negative pathogens, we initiated a study of selected putative Chlamydia T3S chaperones in an effort to gain mechanistic insight into the Chlamydia T3SS and to potentially identify Chlamydia-specific secreted products. C. trachomatis Scc2 and Scc3 are homologous to SycD of Yersinia spp. Functional studies of the heterologous Yersinia T3SS indicated that although neither Scc2 nor Scc3 was able to fully complement a sycD null mutant, both have SycD-like characteristics. Both were able to associate with the translocator protein YopD, and Scc3 expression restored limited secretion of YopD in in vitro studies of T3S. CopB (CT578) and CopB2 (CT861) are encoded adjacent to scc2 and scc3, respectively, and have structural similarities with the YopB family of T3S translocators. Either Scc2 or Scc3 coprecipitates with CopB from C. trachomatis extracts. Expression of CopB or CopB2 in Yersinia resulted in their type III-dependent secretion, and localization studies with C. trachomatis-infected cells indicated that both were secreted by Chlamydia.

Temporal analysis of Coxiella burnetii morphological differentiation.

Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis.

The chlamydial developmental cycle is characterized by an intracellular replicative form, termed the reticulate body, and an extracellular form called the elementary body. Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1. Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active, although the mechanisms of Hc1 release from DNA have remained unknown. Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1. A genetic screen was therefore designed to identify chlamydial genes rescuing Escherichia coli from the lethal effects of Hc1 overexpression. CT804, a gene homologous to ispE, which encodes an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was selected. E. coli coexpressing CT804 and Hc1 grew normally, although they expressed Hc1 to a level equivalent to that which condensed the chromatin of parent Hc1-expressing controls. Inhibition of the MEP pathway with fosmidomycin abolished IspE rescue of Hc1-expressing E. coli. Deproteinated extract from IspE-expressing bacteria caused dispersal of purified chlamydial nucleoids, suggesting that chlamydial histone-DNA interactions are disrupted by a small metabolite within the MEP pathway rather than by direct action of IspE. By partial reconstruction of the MEP pathway, we determined that 2-C-methylerythritol 2,4-cyclodiphosphate dissociated Hc1 from chlamydial chromatin. These results suggest that chlamydial histone-DNA interactions are disrupted upon germination by a small metabolite in the MEP pathway of isoprenoid biosynthesis.

Golgi-dependent transport of cholesterol to the Chlamydia trachomatis inclusion.

Cholesterol, a lipid not normally found in prokaryotes, was identified in purified Chlamydia trachomatis elementary bodies and in the chlamydial parasitophorous vacuole (inclusion) membrane of infected HeLa cells. Chlamydiae obtained eukaryotic host cell cholesterol both from de novo synthesis or low-density lipoprotein. Acquisition of either de novo-synthesized cholesterol or low-density lipoprotein-derived cholesterol was microtubule-dependent and brefeldin A-sensitive, indicating a requirement for the Golgi apparatus. Transport also required chlamydial protein synthesis, indicative of a pathogen-directed process. The cholesterol trafficking pathway appears to coincide with a previously characterized delivery of sphingomyelin to the inclusion in that similar pharmacological treatments inhibited transport of both sphingomyelin and cholesterol. These results support the hypothesis that sphingomyelin and cholesterol may be cotransported via a Golgi-dependent pathway and that the chlamydial inclusion receives cholesterol preferentially from a brefeldin A-sensitive pathway of cholesterol trafficking from the Golgi apparatus to the plasma membrane.