Identification of C(4)-dicarboxylate transport systems in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa utilizes preferentially C(4)-dicarboxylates such as malate, fumarate, and succinate as carbon and energy sources. We have identified and characterized two C(4)-dicarboxylate transport (Dct) systems in P. aeruginosa PAO1. Inactivation of the dctA(PA1183) gene caused a growth defect of the strain in minimal media supplemented with succinate, fumarate or malate, indicating that DctA has a major role in Dct. However, residual growth of the dctA mutant in these media suggested the presence of additional C(4)-dicarboxylate transporter(s). Tn5 insertion mutagenesis of the ΔdctA mutant led to the identification of a second Dct system, i.e., the DctPQM transporter belonging to the tripartite ATP-independent periplasmic (TRAP) family of carriers. The ΔdctA ΔdctPQM double mutant showed no growth on malate and fumarate and residual growth on succinate, suggesting that DctA and DctPQM are the only malate and fumarate transporters, whereas additional transporters for succinate are present. Using lacZ reporter fusions, we showed that the expression of the dctA gene and the dctPQM operon was enhanced in early exponential growth phase and induced by C(4)-dicarboxylates. Competition experiments demonstrated that the DctPQM carrier was more efficient than the DctA carrier for the utilization of succinate at micromolar concentrations, whereas DctA was the major transporter at millimolar concentrations. To conclude, this is the first time that the high- and low-affinity uptake systems for succinate DctA and DctPQM have been reported to function coordinately to transport C(4)-dicarboxylates and that the alternative sigma factor RpoN and a DctB/DctD two-component system regulates simultaneously the dctA gene and the dctPQM operon.

Hepatitis C Virus Nonstructural 5A Protein Inhibits Lipopolysaccharide-Mediated Apoptosis of Hepatocytes by Decreasing Expression of Toll-Like Receptor 4.

Background. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis. The aim of this study was to assess the effects of HCV NS5A on apoptosis induced by Toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Methods. Apoptotic responses to TLR4 ligands and the expression of molecules involved in TLR signaling pathways in human hepatocytes were examined with or without expression of HCV NS5A. Results. HCV NS5A protected HepG2 hepatocytes against LPS-induced apoptosis, an effect linked to reduced TLR4 expression. A similar downregulation of TLR4 expression was observed in Huh-7-expressing genotype 1b and 2a. In agreement with these findings, NS5A inhibited the expression of numerous genes encoding for molecules involved in TLR4 signaling, such as CD14, MD-2, myeloid differentiation primary response gene 88, interferon regulatory factor 3, and nuclear factor-κB2. Consistent with a conferred prosurvival advantage, NS5A diminished the poly(adenosine diphosphate-ribose) polymerase cleavage and the activation of caspases 3, 7, 8, and 9 and increased the expression of anti-apoptotic molecules Bcl-2 and c-FLIP. Conclusions. HCV NS5A downregulates TLR4 signaling and LPS-induced apoptotic pathways in human hepatocytes, suggesting that disruption of TLR4-mediated apoptosis may play a role in the pathogenesis of HCV infection.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Phylogenomics of C(4) photosynthesis in sedges (Cyperaceae): multiple appearances and genetic convergence.

C(4) photosynthesis is an adaptive trait conferring an advantage in warm and open habitats. It originated multiple times and is currently reported in 18 plant families. It has been recently shown that phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the C(4) pathway, evolved through numerous independent but convergent genetic changes in grasses (Poaceae). To compare the genetics of multiple C(4) origins on a broader scale, we reconstructed the evolutionary history of the C(4) pathway in sedges (Cyperaceae), the second most species-rich C(4) family. A sedge phylogeny based on two plastome genes (rbcL and ndhF) has previously identified six fully C(4) clades. Here, a relaxed molecular clock was used to calibrate this tree and showed that the first C(4) acquisition occurred in this family between 19.6 and 10.1 Ma. According to analyses of PEPC-encoding genes (ppc), at least five distinct C(4) origins are present in sedges. Two C(4) Eleocharis species, which were unrelated in the plastid phylogeny, acquired their C(4)-specific PEPC genes from a single source, probably through reticulate evolution or a horizontal transfer event. Acquisitions of C(4) PEPC in sedges have been driven by positive selection on at least 16 codons (3.5% of the studied gene segment). These sites underwent parallel genetic changes across the five sedge C(4) origins. Five of these sites underwent identical changes also in grass and eudicot C(4) lineages, indicating that genetic convergence is most important within families but that identical genetic changes occurred even among distantly related taxa. These lines of evidence give new insights into the constraints that govern molecular evolution.

In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease.

In contrast to intact BALB/c mice, BALB/c mice rendered deficient in Vbeta4+ CD4+ T cells develop a Th1 response to infection with Leishmania major and are resistant. Vbeta4-deficient BALB/c mice are unable to generate the early IL-4 transcription occurring in Vbeta4 Valpha8 CD4+ T cells of BALB/c mice within 1 day of infection. Here we demonstrate that treatment of Vbeta4-deficient BALB/c mice with IL-4 during the first 64 h after infection instructs Th2 cell development and susceptibility to infection. The demonstrated inability of IL-4 to reverse the resistant phenotype of BALB/c mice treated with anti-CD4 mAb the day before infection suggest that these effects of IL-4 require its interaction with CD4+ T cells. In contrast to draining lymph node cells from BALB/c mice, cells from Vbeta4-deficient BALB/c mice remain responsive to IL-12 following infection. Strikingly, administration of IL-4 to Vbeta4-deficient BALB/c mice renders their lymph node cells unresponsive to IL-12 by down-regulating IL-12R beta2-chain expression. This study directly demonstrates that in BALB/c mice IL-4 is necessary and sufficient to initiate the molecular events steering Th2 cell maturation and susceptibility to L. major.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells.

Susceptibility and development of Th2 cells in BALB/c mice infected with Leishmania major result from early IL-4 production by Vbeta4Valpha8 CD4+ T cells in response to the Leishmania homolog of mammalian RACK1 Ag. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. We further showed that transfer of 10(7) BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10(7) control BALB/c spleen cells were resistant to infection with L. major and developed a Th1 response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.