Action Spectra of Zebrafish Cone Photoreceptors

Zebrafish is becoming an increasingly popular model in the field of visual neuroscience. Although the absorption spectra of its cone photopigments have been described, the cone action spectra were still unknown. In this study we report the action spectra of the four types of zebrafish cone photoreceptors, determined by measuring voltage responses upon light stimulation using whole cell patch clamp recordings. A generic template of photopigment absorption spectra was fit to the resulting action spectra in order to establish the maximum absorption wavelength, the A2-based photopigment contribution and the size of the β-wave of each cone-type. Although in general there is close correspondence between zebrafish cone action- and absorbance spectra, our data suggest that in the case of MWS- and LWS-cones there is appreciable contribution of A2-based photopigments and that the β-wave for these cones is smaller than expected based on the absorption spectra.     

Anti-malarial drug targets: Screening for inhibitors of 2C-methyl-d-erythritol 4-phosphate synthase (IspC protein) in Mediterranean plants

The recently discovered non-mevalonate pathway of isoprenoid biosynthesis serves as the unique source of terpenoids in numerous pathogenic eubacteria and in apicoplast-type protozoa, most notably Plasmodium, but is absent in mammalian cells. It is therefore an attractive target for anti-infective chemotherapy. The first committed step of the non-mevalonate pathway is catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase (IspC). Using photometric and NMR spectroscopic assays, we screened extracts of Mediterranean plants for inhibitors of the enzyme. Strongest inhibitory activity was found in leaf extracts of Cercis siliquastrum.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Biosynthesis of isoprenoids: studies on the mechanism of 2C-methyl-D-erythritol-4-phosphate synthase

2C-Methyl-D-erythritol-4-phosphate synthase, encoded by the ispC gene (also designated dxr), catalyzes the first committed step in the nonmevalonate isoprenoid biosynthetic pathway. The reaction involves the isomerization of 1-deoxy-D-xylulose 5-phosphate, giving a branched-chain aldose derivative that is subsequently reduced to 2C-methyl-D-erythritol 4-phosphate. The isomerization step has been proposed to proceed as an intramolecular rearrangement or a retroaldol-aldol sequence. We report the preparation of (13)C-labeled substrate isotopologs that were designed to optimize the detection of an exchange of putative cleavage products that might occur in the hypothetical retroaldol-aldol reaction sequence. In reaction mixtures containing large amounts of 2C-methyl-D-erythritol-4-phosphate synthase from Escherichia coli, Mycobacterium tuberculosis or Arabidopsis thaliana, and a mixture of [1-(13)C(1)]-2C-methyl-D-erythritol 4-phosphate and [3-(13)C(1)]2C-methyl-D-erythritol 4-phosphate, the reversible reaction could be followed over thousands of reaction cycles. No fragment exchange could be detected by NMR spectroscopy, and the frequency of exchange, if any, is less than 5 p.p.m. per catalytic cycle. Hydroxyacetone, the putative second fragment expected from the retroaldol cleavage, was not incorporated into the enzyme product. In contrast to other reports, IspC did not catalyze the isomerisation of 1-deoxy-D-xylulose 5-phosphate to give 1-deoxy-L-ribulose 5-phosphate under any conditions tested. However, we could show that the isomerization reaction proceeds at room temperature without a requirement for enzyme catalysis. Although a retroaldol-aldol mechanism cannot be ruled out conclusively, the data show that a retroldol-aldol reaction sequence would have to proceed with very stringent fragment containment that would apply to the enzymes from three genetically distant organisms.     

Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²⁺ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.     

Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC₅₀ of 4 μm (K_i = 1 μm). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-¹³C₆]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [¹³C₃]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC^- mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1^- and YscM2^- mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a “load-and-shoot cycle” model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.Type three secretion systems (T3SSs)³ are used by several Gram-negative bacteria as microinjection devices to deliver effector proteins into host cells (1). The translocated effector proteins reprogram the host cell in favor of the microbial invader or symbiont. Pathogenic yersiniae (the enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis and the plague bacillus Yersinia pestis) utilize a plasmid-encoded T3SS to undermine the host primary immune response (2). This is mediated by the injection of a set of effector proteins called Yops (Yersinia outer proteins) into host cells, in particular into cells with innate immune functions, such as macrophages, dendritic cells, and neutrophils (3). The concerted action of Yops, targeting multiple signaling pathways, results in actin cytoskeleton disruption, suppression of proinflammatory signaling, and induction of apoptosis. This strategy enables yersiniae to multiply extracellularly in host tissue.Expression of the Yersinia T3SS is up-regulated at 37 °C, and translocation of Yops across the host cell membrane is triggered by cell contact (4, 5). Pathogenic yersiniae cultivated under low calcium conditions at 37 °C express a phenotype referred to as “low calcium response” (LCR). The LCR is characterized by growth restriction as well as massive expression and secretion of Yops into the culture medium (6–9). The allocation of energy and metabolites for the massive synthesis and transport of Yops is demanding, and this burden is believed to be responsible for the observed growth inhibition (10). To give an idea of the metabolic requirements, Yops are secreted to the culture supernatant in 10-mg amounts per liter of culture within 2 h after calcium depletion of the medium. Furthermore, post-translationally secreted substrates need to be unfolded by a T3SS-specific ATPase prior to secretion (11–14). In addition, T3SS-dependent transport of Yops requires the proton motive force (15). However, there is evidence that growth cessation and Yop expression can be uncoupled (16, 17), suggesting a coordinated regulation of metabolism and protein transport rather than the LCR reflecting an inevitable physiological consequence.What are the candidate proteins that could be involved in such a coordination? YscM1 and YscM2 (57% identical to YscM1) are key candidates, since they act at a major nodal point of the T3SS regulatory network in Y. enterocolitica. In Y. pestis and Y. pseudotuberculosis, only the YscM1 homologue LcrQ exists (99% identical to YscM1). YscM1/LcrQ and YscM2 are secretion substrates of the T3SS that are involved in up-regulation of Yop expression after host cell contact. Upon cell contact, the decrease of intracellular levels of YscM1/LcrQ and YscM2 due to their translocation into host cells results in a derepression of Yop synthesis (18–21). The two yscM copies of Y. enterocolitica were presumed to be functionally equivalent, since deletion of either gene was found to be phenotypically silent (19, 22). Only deletion of both yscM genes could establish the lcrQ phenotype (19, 22), distinguished by temperature sensitivity for growth, derepressed Yop expression, and secretion of LcrV and YopD in the presence of calcium ions.YscM1/LcrQ as well as YscM2 exhibit homology to the N terminus of the effector YopH (19, 23, 24), a fact that may explain their shared assistance by SycH (specific Yop chaperone) (20, 25). It was shown that YscM1/LcrQ and YscM2 exert their influence on Yop expression in concert with the T3SS components SycH, SycD (LcrH in Y. pestis and Y. pseudotuberculosis), and YopD (21, 26–28). It is further described that YscM1 and/or YscM2 interact with several of the T3SS-specific chaperones, in particular with SycH, SycE, SycD, and SycO (20, 29–31). This has led to the model that YscM/LcrQ proteins might function as an interface that senses whether chaperones are loaded with Yops and transduces these signals into control of Yop expression (14).These features of YscM1/LcrQ and YscM2 prompted us to speculate about a key role of these proteins in coordination of metabolism and expression of T3SS components. Using recombinant GST-YscM1 and GST-YscM2 as bait for Y. enterocolitica cytosolic proteins, we identified phosphoenolpyruvate carboxylase (PEPC) as interaction partner of both YscM1 and YscM2. Under in vitro conditions, YscM1 down-regulated PEPC activity and bacterial growth/replication. Isotopologue profiling of Yop proteins and derived amino acids from Y. enterocolitica grown in the presence of [U-¹³C₆]glucose showed the functionality of the PEPC reaction under virulence conditions (isotopologues are molecular entities that differ only in isotopic composition (number of isotopic substitutions); e.g. CH₄, CH₃D, and CH₂D₂). Moreover, biosynthetic rates of amino acids were increased in mutants defective in YscM1 or YscM2, suggesting a general role of these regulators in the metabolism of Y. enterocolitica. Recently, evidence has been accumulating that the metabolic state contributes to the regulation of T3SSs of diverse pathogens, also including the flagellar T3SS in Pseudomonas and Salmonella (32–36).     

Recent advances in the diagnosis and treatment of hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening disease of severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and macrophages secreting high amounts of inflammatory cytokines. It is a frequent manifestation in patients with predisposing genetic defects, but can occur secondary to various infectious, malignant, and autoimmune triggers in patients without a known genetic predisposition. Clinical hallmarks are prolonged fever, cytopenias, hepatosplenomegaly, and neurological symptoms, but atypical variants presenting with signs of chronic immunodeficiency are increasingly recognized. Impaired secretion of perforin is a key feature in several genetic forms of the disease, but not required for disease pathogenesis. Despite progress in diagnostics and therapy, mortality of patients with severe HLH is still above 40%. Reference treatment is an etoposide-based protocol, but new approaches are currently explored. Key for a favorable prognosis is the rapid identification of an underlying genetic cause, which has been facilitated by recent immunological and genetic advances. In patients with predisposing genetic disease, hematopoietic stem cell transplantation is performed increasingly with reduced intensity conditioning regimes. Current research aims at a better understanding of disease pathogenesis and evaluation of more targeted approaches to therapy, including anti-cytokine antibodies and gene therapy.     

IL-18 improves the early antimicrobial host response to pneumococcal pneumonia.

To determine the role of endogenous IL-18 during pneumonia, IL-18 gene-deficient (IL-18(-/-)) mice and wild-type (WT) mice were intranasally inoculated with Streptococcus pneumoniae, the most common causative agent of community-acquired pneumonia. Infection with S. pneumoniae increased the expression of IL-18 mRNA and was associated with elevated concentrations of both precursor and mature IL-18 protein within the lungs. IL-18(-/-) mice had significantly more bacteria in their lungs and were more susceptible for progressing to systemic infection at 24 and 48 h postinoculation. Similarly, treatment of WT mice with anti-IL-18 was associated with enhanced outgrowth of pneumococci. In contrast, the clearance of pneumococci from lungs of IL-12(-/-) mice was unaltered when compared with WT mice. Furthermore, anti-IL-12 did not influence bacterial clearance in either IL-18(-/-) or WT mice. These data suggest that endogenous IL-18, but not IL-12, plays an important role in the early antibacterial host response during pneumococcal pneumonia.