Transposon Mutagenesis in Chlamydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Lateral Gene Transfer

Transposon mutagenesis is a widely applied and powerful genetic tool for the discovery of genes associated with selected phenotypes. Chlamydia trachomatis is a clinically significant, obligate intracellular bacterium for which many conventional genetic tools and capabilities have been developed only recently. This report describes the successful development and application of a Himar transposon mutagenesis system for generating single-insertion mutant clones of C. trachomatis. This system was used to generate a pool of 105 transposon mutant clones that included insertions in genes encoding flavin adenine dinucleotide (FAD)-dependent monooxygenase (C. trachomatis 148 [ct148]), deubiquitinase (ct868), and competence-associated (ct339) proteins. A subset of Tn mutant clones was evaluated for growth differences under cell culture conditions, revealing that most phenocopied the parental strain; however, some strains displayed subtle and yet significant differences in infectious progeny production and inclusion sizes. Bacterial burden studies in mice also supported the idea that a FAD-dependent monooxygenase (ct148) and a deubiquitinase (ct868) were important for these infections. The ct339 gene encodes a hypothetical protein with limited sequence similarity to the DNA-uptake protein ComEC. A transposon insertion in ct339 rendered the mutant incapable of DNA acquisition during recombination experiments. This observation, along with in situ structural analysis, supports the idea that this protein is playing a role in the fundamental process of lateral gene transfer similar to that of ComEC. In all, the development of the Himar transposon system for Chlamydia provides an effective genetic tool for further discovery of genes that are important for basic biology and pathogenesis aspects.S.D.L., Z.E.D., K.S.H., S.B., R.J.S., and P.S.H. were funded by NIH (AI126785)J.W. and P.S.H. were supported by NIH AI125929. P.S.H. was also supported by P20GM113117Support for genomic sequencing was supplemented by P20GM10363

Structure of a bacterial type III secretion system in contact with a host membrane in situ

Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform– ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action’ conformational changes that underpin effector injection

Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l-Rhamnose-Responsive rhaSR Promoter in Escherichia coli

The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position −111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (α-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the α-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position

Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes▿

The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2

Coactivating Signals for the Hepatic Lymphocyte Gamma Interferon Response to Francisella tularensis

The facultative intracellular bacterium Francisella tularensis is capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site of F. tularensis infection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-γ), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-γ when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type A Francisella tularensis subsp. tularensis organisms were comparable to the live attenuated vaccine strain of Francisella tularensis subsp. holarctica in their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses to F. tularensis are resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses

Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain▿

The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS

Francisella tularensis Induces Extensive Caspase-3 Activation and Apoptotic Cell Death in the Tissues of Infected Mice▿

Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80+ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen