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David S Weiss

Publications and source records attributed to David S Weiss.

At least 19 recordsLinked to original sources
PubMed2006

Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis.

The facultative intracellular bacterium Francisella tularensis causes the zoonotic disease tularemia. F. tularensis resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several Francisella genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the Francisella genome by transcriptional profiling of wild-type and mglA mutant bacteria would lead to the discovery of new virulence factors utilized by F. tularensis. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the Francisella pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (pdpB and cds2) as well as outside the FPI (FTT0989, oppB, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new Francisella virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.

Animals↗
PubMed2006

Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor.

Picrotoxin, a potent antagonist of the inhibitory central nervous system GABAA and glycine receptors, is believed to interact with residues that line the central ion pore. These pore-lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6' location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the alpha, beta or gamma subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two alpha subunits, two beta subunits and one gamma subunit, it is not clear how many alpha and beta subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant alpha or beta subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6' mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.

Animals↗
PubMed2006

Mechanism of action of benzodiazepines on GABAA receptors.

Wild-type and mutant alpha1beta2gamma2 GABA(A) receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. Dose-response relationships for GABA were compared in the absence and presence of 1 microM diazepam (DZP) or methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM). The dose-current relationships yielded EC(50)'s (concentration for half-maximal activation) of 41.0+/-3.0, 21.7+/-2.7, and 118.3+/-6.8 microM for GABA, GABA plus DZP, and GABA plus DMCM, respectively.DZP- and DMCM-mediated modulation were examined in GABA(A) receptors in which the beta-subunit carries the L259S mutation. This mutation has been shown to produce spontaneous opening and impart a leftward shift in the dose-response relationship. In this case, neither DZP nor DMCM produced a significant alteration in the GABA dose-response relationship with GABA EC(50)'s of 0.078+/-0.005, 0.12+/-0.03, and 0.14+/-0.004 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM.DZP- and DMCM-mediated modulations were examined in GABA(A) receptors in which the alpha-subunit carries the L263S mutation. This mutation also produced spontaneous opening and a leftward shift of the GABA dose-response relation, but to a lesser extent than that of betaL259S. In this case, the leftward and rightward shifts for DZP and DMCM were still present with EC(50)'s=0.24+/-0.03, 0.14+/-0.02, and 1.2+/-0.04 microM for GABA, GABA plus 1 microM DZP, and GABA plus 1 microM DMCM, respectively.Oocytes expressing ultrahigh levels of wild-type GABA(A) receptors exhibited currents in response to 1 muM DZP alone, whereas DMCM decreased the baseline current. The DZP-mediated activation currents were determined in wild-type receptors as well as receptors in which the GABA binding site was mutated (beta2Y205S). The EC(50)'s for DZP-mediated activation were 72.0+/-2.0 and 115+/-6.2 nM, respectively, similar to the EC(50) for DZP-mediated enhancement of the wild-type GABA-activated current (64.8+/-3.7 nM). Our results support a mechanism in which DZP increases the apparent affinity of the receptor, not by altering the affinity of the closed state, but rather by shifting the equilibrium towards the high-affinity open state.

Algorithms↗
PubMed2006

A quantum Newton's cradle.

It is a fundamental assumption of statistical mechanics that a closed system with many degrees of freedom ergodically samples all equal energy points in phase space. To understand the limits of this assumption, it is important to find and study systems that are not ergodic, and thus do not reach thermal equilibrium. A few complex systems have been proposed that are expected not to thermalize because their dynamics are integrable. Some nearly integrable systems of many particles have been studied numerically, and shown not to ergodically sample phase space. However, there has been no experimental demonstration of such a system with many degrees of freedom that does not approach thermal equilibrium. Here we report the preparation of out-of-equilibrium arrays of trapped one-dimensional (1D) Bose gases, each containing from 40 to 250 (87)Rb atoms, which do not noticeably equilibrate even after thousands of collisions. Our results are probably explainable by the well-known fact that a homogeneous 1D Bose gas with point-like collisional interactions is integrable. Until now, however, the time evolution of out-of-equilibrium 1D Bose gases has been a theoretically unsettled issue, as practical factors such as harmonic trapping and imperfectly point-like interactions may compromise integrability. The absence of damping in 1D Bose gases may lead to potential applications in force sensing and atom interferometry.

Journal Article↗
PubMed2006

Cryopyrin activates the inflammasome in response to toxins and ATP.

A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.

Adenosine Triphosphate↗
PubMed2005

Local pair correlations in one-dimensional Bose gases.

We measure photoassociation rates in one-dimensional Bose gases, and so determine the local pair correlation function over a wide range of coupling strengths. As bosons become more strongly coupled, we observe a tenfold decrease in their wave function overlap, thus directly observing the fermionization of bosons.

Journal Article↗
PubMed2005

Innate immunity against Francisella tularensis is dependent on the ASC/caspase-1 axis.

Francisella tularensis is a highly infectious gram-negative coccobacillus that causes the zoonosis tularemia. This bacterial pathogen causes a plague-like disease in humans after exposure to as few as 10 cells. Many of the mechanisms by which the innate immune system fights Francisella are unknown. Here we show that wild-type Francisella, which reach the cytosol, but not Francisella mutants that remain localized to the vacuole, induced a host defense response in macrophages, which is dependent on caspase-1 and the death-fold containing adaptor protein ASC. Caspase-1 and ASC signaling resulted in host cell death and the release of the proinflammatory cytokines interleukin (IL)-1beta and IL-18. F. tularensis-infected caspase-1- and ASC-deficient mice showed markedly increased bacterial burdens and mortality as compared with wild-type mice, demonstrating a key role for caspase-1 and ASC in innate defense against infection by this pathogen.

Animals↗
PubMed2005

A deficiency in S-adenosylmethionine synthetase interrupts assembly of the septal ring in Escherichia coli K-12.

A mutant in which S-adenosylmethionine synthetase is underexpressed makes filaments with no visible septa. Examination with GFP fusions to various septal proteins shows that FtsZ, ZipA and FtsA localize to the septal ring, but FtsQ, FtsW, FtsI or FtsN do not. The requirement for S-adenosylmethionine suggests that some methylation reaction is required before a complete septal ring can be assembled.

Anti-Bacterial Agents↗
PubMed2005

MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus.

It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88(-/-) mice do not clear the bacteria as efficiently as wild-type, TLR4(-/-), TLR2(-/-), or TLR4/TLR2(-/-) mice.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The transmembrane helix of the Escherichia coli division protein FtsI localizes to the septal ring.

FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is unusual [corrected] for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.

Amino Acid Sequence↗
PubMed2005

The lipopolysaccharide of Brucella abortus BvrS/BvrR mutants contains lipid A modifications and has higher affinity for bactericidal cationic peptides.

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic beta(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants.

Acylation↗
PubMed2004

Mapping the rho1 GABA(C) receptor agonist binding pocket. Constructing a complete model.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA(C)) is a ligand-gated ion channel with pharmacological properties distinct from the GABA(A) receptor. To date, only three binding domains in the recombinant rho1 GABA(C) receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the rho1 GABA(C) receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA(C) receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a beta-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the rho1 GABA(C) receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA(C) receptor binding pocket was proposed and discussed in comparison with the GABA(A) receptor binding pocket.

Amino Acid Sequence↗
PubMed2004

Picrotoxin-mediated antagonism of alpha3beta4 and alpha7 acetylcholine receptors.

Picrotoxin (PTX) is a convulsant that antagonizes many inhibitory ligand-gated receptors. The mechanism of PTX block is believed to involve residues which line the pore in the second transmembrane domain (M2). The alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (nAChRs) have high homology to inhibitory LGICs in this M2 region and therefore could also be susceptible to block by PTX. Here, we report that PTX is an effective inhibitor at these nicotinic receptors (rat), with IC50 values of 96.1 +/- 5.5 and 194.9 +/- 19.2 microM for the alpha(3)beta(4) and alpha(7), respectively. These results provide insights into the structure-function relation of PTX-mediated antagonism in this family of ligand-activated receptors. Furthermore they should also be considered when employing PTX to selectively eliminate GABA- or glycine-mediated events.

Animals↗
PubMed2004

Observation of a one-dimensional Tonks-Girardeau gas.

We report the observation of a one-dimensional (1D) Tonks-Girardeau (TG) gas of bosons moving freely in 1D. Although TG gas bosons are strongly interacting, they behave very much like noninteracting fermions. We enter the TG regime with cold rubidium-87 atoms by trapping them with a combination of two light traps. By changing the trap intensities, and hence the atomic interaction strength, the atoms can be made to act either like a Bose-Einstein condensate or like a TG gas. We measure the total 1D energy and the length of the gas. With no free parameters and over a wide range of coupling strengths, our data fit the exact solution for the ground state of a 1D Bose gas.

Journal Article↗
PubMed2004

Toll-like receptors are temporally involved in host defense.

Toll-like receptors (TLRs) are evolutionarily conserved proteins that recognize microbial molecules and initiate host defense. To investigate how TLRs work together to fight infections, we tested the role of TLRs in host defense against the Gram-negative bacterial pathogen, Salmonella. We show that TLR4 is critical for early cytokine production and killing of bacteria by murine macrophages. Interestingly, later on, TLR2, but not TLR4, is required for macrophage responses. Myeloid differentiation factor 88, an adaptor protein directly downstream of TLRs, is required for both early and late responses. TLR4, TLR2, and myeloid differentiation factor 88 are involved in murine host defense against Salmonella in vivo, which correlates with the defects in host defense observed in vitro. We propose a model where the sequential activation of TLRs tailors the immune response to different microbes.

Acute Disease↗
PubMed2004

Evidence that the TM1-TM2 loop contributes to the rho1 GABA receptor pore.

Considerable evidence indicates the second transmembrane domain (TM2) of the gamma-aminobutyric acid (GABA) receptor lines the integral ion pore. To further delineate the structures that constitute the ion pore and selectivity filter of the rho1 GABA receptor, we used the substituted cysteine accessibility method with charged reagents to identify anion- and cation-accessible surfaces. Twenty-one consecutive residues were mutated to cysteine, one at a time, in the presumed intracellular end of the first transmembrane domain (TM1; Ala(271)-Met(276)), the entire linker connecting TM1 to TM2 (Leu(277)-Arg(287)), and the presumed intracellular end of TM2 (Ala(288)-Ala(291)). Positively (MTSEA(+)) and negatively (pCMBS(-)) charged sulfhydryl reagents, as well as Cd(2+), were added extracellularly to test accessibility of the engineered cysteines. Four of the mutants, all at the intracellular end of TM2 (R287C, V289C, P290C, A291C), were accessible to positively charged reagents, whereas seven mutants (A271C, T272C, L277C, W279C, V280C, P290C, A291C) were functionally modified by negatively charged pCMBS(-). These seven modified residues were at the intracellular end of TM2, in the TM1-TM2 linker, and at the intracellular end of TM1. In nearly all cases (excluding P290C), the rate and the degree of modification were state-dependent, with greater accessibility in the presence of agonist. Select cysteine mutants were combined with a point mutation (A291E) that converted the pore from chloride- to non-selective. In this case, positively charged reagents could modify residues in the TM1-TM2 linker (Leu(277) and Val(280)), supporting the notion that the modifying reagents were reaching their target through the pore. Taken together, our results suggest that, up to its intracellular end, the TM2 domain is not charge selective. In addition, we propose that the TM1-TM2 linker and the intracellular end of TM1 are along the pathway of the permeating ion. These findings may lend new insights into the structure of the GABA receptor pore.

Amino Acid Sequence↗
PubMed2004

Neutrophil extracellular traps kill bacteria.

Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.

Animals↗