STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection.

UnlabelledSTING (stimulator of interferon [IFN] genes) initiates type I IFN responses in mammalian cells through the detection of microbial nucleic acids. The membrane-bound obligate intracellular bacterium Chlamydia trachomatis induces a STING-dependent type I IFN response in infected cells, yet the IFN-inducing ligand remains unknown. In this report, we provide evidence that Chlamydia synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite not previously identified in Gram-negative bacteria, and that this metabolite is a prominent ligand for STING-mediated activation of IFN responses during infection. We used primary mouse lung fibroblasts and HEK293T cells to compare IFN-β responses to Chlamydia infection, c-di-AMP, and other type I IFN-inducing stimuli. Chlamydia infection and c-di-AMP treatment induced type I IFN responses in cells expressing STING but not in cells expressing STING variants that cannot sense cyclic dinucleotides but still respond to cytoplasmic DNA. The failure to induce a type I IFN response to Chlamydia and c-di-AMP correlated with the inability of STING to relocalize from the endoplasmic reticulum to cytoplasmic punctate signaling complexes required for IFN activation. We conclude that Chlamydia induces STING-mediated IFN responses through the detection of c-di-AMP in the host cell cytosol and propose that c-di-AMP is the ligand predominantly responsible for inducing such a response in Chlamydia-infected cells.ImportanceThis study shows that the Gram-negative obligate pathogen Chlamydia trachomatis, a major cause of pelvic inflammatory disease and infertility, synthesizes cyclic di-AMP (c-di-AMP), a nucleic acid metabolite that thus far has been described only in Gram-positive bacteria. We further provide evidence that the host cell employs an endoplasmic reticulum (ER)-localized cytoplasmic sensor, STING (stimulator of interferon [IFN] genes), to detect c-di-AMP synthesized by Chlamydia and induce a protective IFN response. This detection occurs even though Chlamydia is confined to a membrane-bound vacuole. This raises the possibility that the ER, an organelle that innervates the entire cytoplasm, is equipped with pattern recognition receptors that can directly survey membrane-bound pathogen-containing vacuoles for leaking microbe-specific metabolites to mount type I IFN responses required to control microbial infections

Toward community standards in the quest for orthologs

The identification of orthologs—genes pairs descended from a common ancestor through speciation, rather than duplication—has emerged as an essential component of many bioinformatics applications, ranging from the annotation of new genomes to experimental target prioritization. Yet, the development and application of orthology inference methods is hampered by the lack of consensus on source proteomes, file formats and benchmarks. The second ‘Quest for Orthologs' meeting brought together stakeholders from various communities to address these challenges. We report on achievements and outcomes of this meeting, focusing on topics of particular relevance to the research community at large. The Quest for Orthologs consortium is an open community that welcomes contributions from all researchers interested in orthology research and applications. Contact: [email protected]

Formate Promotes Shigella Intercellular Spread and Virulence Gene Expression

Shigella is an intracellular pathogen that invades the human host cell cytosol and exploits intracellular nutrients for growth, enabling the bacterium to create its own metabolic niche. For Shigella to effectively invade and replicate within the host cytoplasm, it must sense and adapt to changing environmental conditions; however, the mechanisms and signals sensed by S. flexneri are largely unknown. We have found that the secreted Shigella metabolism by-product formate regulates Shigella intracellular virulence gene expression and its ability to spread among epithelial cells. We propose that Shigella senses formate accumulation in the host cytosol as a way to determine intracellular Shigella density and regulate secreted virulence factors accordingly, enabling spatiotemporal regulation of effectors important for dampening the host immune response.The intracellular human pathogen Shigella flexneri invades the colon epithelium, replicates to high cell density within the host cell, and then spreads to adjacent epithelial cells. When S. flexneri gains access to the host cytosol, the bacteria metabolize host cytosolic carbon using glycolysis and mixed acid fermentation, producing formate as a by-product. We show that S. flexneri infection results in the accumulation of formate within the host cell. Loss of pyruvate formate lyase (PFL; ΔpflB), which converts pyruvate to acetyl coenzyme A (CoA) and formate, eliminates S. flexneri formate production and reduces the ability of S. flexneri to form plaques in epithelial cell monolayers. This defect in PFL does not decrease the intracellular growth rate of S. flexneri; rather, it affects cell-to-cell spread. The S. flexneri ΔpflB mutant plaque defect is complemented by supplying exogenous formate; conversely, deletion of the S. flexneri formate dehydrogenase gene fdnG increases host cell formate accumulation and S. flexneri plaque size. Furthermore, exogenous formate increases plaque size of the wild-type (WT) S. flexneri strain and promotes S. flexneri cell-to-cell spread. We also demonstrate that formate increases the expression of S. flexneri virulence genes icsA and ipaJ. Intracellular S. flexneriicsA and ipaJ expression is dependent on the presence of formate, and ipaJ expression correlates with S. flexneri intracellular density during infection. Finally, consistent with elevated ipaJ, we show that formate alters S. flexneri-infected host interferon- and tumor necrosis factor (TNF)-stimulated gene expression. We propose that Shigella-derived formate is an intracellular signal that modulates virulence in response to bacterial metabolism

Intracellular Concentrations of Borrelia burgdorferi Cyclic Di-AMP Are Not Changed by Altered Expression of the CdaA Synthase.

The second messenger nucleotide cyclic diadenylate monophosphate (c-di-AMP) has been identified in several species of Gram positive bacteria and Chlamydia trachomatis. This molecule has been associated with bacterial cell division, cell wall biosynthesis and phosphate metabolism, and with induction of type I interferon responses by host cells. We demonstrate that B. burgdorferi produces a c-di-AMP synthase, which we designated CdaA. Both CdaA and c-di-AMP levels are very low in cultured B. burgdorferi, and no conditions were identified under which cdaA mRNA was differentially expressed. A mutant B. burgdorferi was produced that expresses high levels of CdaA, yet steady state borrelial c-di-AMP levels did not change, apparently due to degradation by the native DhhP phosphodiesterase. The function(s) of c-di-AMP in the Lyme disease spirochete remains enigmatic

Methods for detecting Zika virus in feces: A case study in captive squirrel monkeys (Saimiri boliviensis boliviensis).

A strain of Zika virus (ZIKV) of Asian origin associated with birth defects and neurological disorders has emerged and spread through the Americas. ZIKV was first isolated in the blood of nonhuman primates in Africa and has been detected in the blood, saliva, and urine of a few catarrhine species in both Africa and Asia, suggesting that nonhuman primates may serve as both a source and a reservoir of the virus. The recent introduction of ZIKV to human populations in the Americas presents the potential for the virus to spread into nonhuman primate reservoirs. Thus, it is critical to develop efficient and noninvasive detection methods to monitor the spread of the virus in wild nonhuman primate populations. Here, we describe a method for ZIKV detection in noninvasively collected fecal samples of a Neotropical primate. Fecal samples were collected from two captive squirrel monkeys (Saimiri boliviensis boliviensis) that were experimentally infected with ZIKV (Strain Mexico_1_44) and an additional two uninfected squirrel monkeys. Nucleic acids were extracted from these samples, and RT-qPCR was used to assay for the presence of ZIKV using primers flanking a 101 bp region of the NS5 gene. In both ZIKV-inoculated animals, ZIKV was detected 5-11 days post-infection, but was not detected in the uninfected animals. We compare the fecal results to ZIKV detection in serum, saliva, and urine samples from the same individuals. Our results indicate that fecal detection is a cost-effective, noninvasive method for monitoring wild populations of Neotropical primates as possible ZIKV reservoirs

<i>B</i>. <i>burgdorferi</i> CdaA synthesizes c-di-AMP.

<p>Representative mass spectrometric analysis of cytoplasmic extract from IPTG-induced <i>E</i>. <i>coli</i> strain CRS-0, which expresses <i>B</i>. <i>burgdorferi</i> CdaA from a chimeric plasmid. The identity of the peak at 3.35 min was not determined.</p

Effects of hyper-expressing CdaA in <i>B</i>. <i>burgdorferi</i>.

<p><b>A.</b> Measurements of <i>B</i>. <i>burgdorferi</i> cytoplasmic c-di-AMP levels in samples of uninduced and induced AG1. Bacteria were cultured to mid-exponential phase (approximately 10⁷ bacteria/ml), divided equally divided into two tubes, then <i>cdaA</i> transcription was induced by addition of 0.5 μg/ml (final concentration) anhydrotetracycline (ATc) to one tube, and both were incubated for 24h at 35°C. Equal volumes of borrelial cell extracts were analyzed. <b>B.</b> Immunoblot analyses of KS50 and AG1, without and with inclusion of 0.5 μg/ml anhydrotetracycline (ATc) inducer (- and +, respectively). Membranes were probed with antibodies directed against CdaA or the constitutively-expressed FlaB subunit of the flagella. Wild-type and uninduced AG1 bacteria produced substantially less CdaA than did induced AG1, and the immunoblot signal was not detectable for those strains/conditions at the illustrated exposure. Analyses of mRNA levels also indicated that <i>cdaA</i> is expressed at low levels by uninduced AG1 (data not shown). <b>C and D.</b> Q-RT-PCR analyses of the effects of CdaA hyperexpression on transcription of select <i>B</i>. <i>burgdorferi</i> mRNAs. Transcript fold changes are shown as the difference between uninduced and induced cultures for both strains KS50 and AG1, relative to control <i>flaB</i> or <i>recA</i>, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125440#pone.0125440.ref030" target="_blank">30</a>]. Multiple t tests were performed for each strain and examined transcript. Only the differences in levels of <i>cdaA</i> transcripts in induced cultures of AG1 were significant (indicated by **, p = 0.0012 when compared with <i>flaB</i>, and p = 0.0023 when compared with <i>recA</i>).</p

Alignment of the predicted amino acid sequences of <i>B</i>. <i>burgdorferi</i> CdaA and closely-related ci-di-AMP synthases of other bacteria.

<p>The two regions of conserved residues that constitute the DAC domain are boxed in blue. Residues found in all 5 proteins are indicated by an asterisk (*), residues in 4 proteins by a colon (:), and those in 3 proteins by a period (.). Enzyme sequences are identified as: Bb, <i>B</i>. <i>burgdorferi</i> CdaA; Bs, <i>Bacillus subtilis</i> CdaA (formerly YbbP); Lm, <i>Listeria monocytogenes</i> CdaA/DacA; Sa, <i>Staphylococcus aureus</i> DacA; and Ct, <i>C</i>. <i>trachomatis</i> DacA.</p