Inflammasome Activation Triggers Blood Clotting and Host Death through Pyroptosis.

Inflammasome activation and subsequent pyroptosis are critical defense mechanisms against microbes. However, overactivation of inflammasome leads to death of the host. Although recent studies have uncovered the mechanism of pyroptosis following inflammasome activation, how pyroptotic cell death drives pathogenesis, eventually leading to death of the host, is unknown. Here, we identified inflammasome activation as a trigger for blood clotting through pyroptosis. We have shown that canonical inflammasome activation by the conserved type III secretion system (T3SS) rod proteins from Gram-negative bacteria or noncanonical inflammasome activation by lipopolysaccharide (LPS) induced systemic blood clotting and massive thrombosis in tissues. Following inflammasome activation, pyroptotic macrophages released tissue factor (TF), an essential initiator of coagulation cascades. Genetic or pharmacological inhibition of TF abolishes inflammasome-mediated blood clotting and protects against death. Our data reveal that blood clotting is the major cause of host death following inflammasome activation and demonstrate that inflammasome bridges inflammation with thrombosis.

Emergence of two AcrB substitutions conferring multidrug resistance to Salmonella spp.

AcrAB-TolC is a major tripartite multidrug efflux pump conferring resistance to a wide variety of compounds in Gram-negative pathogens. Many AcrB mutants have been constructed through site-directed mutagenesis to probe the mechanism of AcrB function in antibiotic resistance. However, much less is known about the actual drug resistance related mutants that naturally occur in clinically isolated pathogens. Here, we report two novel AcrB substitutions, M78I and P319L, in clinically isolated Salmonella strains with high-level ciprofloxacin resistance. Plasmids expressing the detected acrB mutations were constructed and introduced into SL1344△acrB Antimicrobial susceptibility assay showed that all AcrB M78I, AcrB P319L and AcrB M78I/319L conferred reduced susceptibilities to multiple substrates, including fluoroquinolones, erythromycin, tetracyclines, bile salts and dyes. Site-directed mutagenesis and MIC results revealed that increased hydrophobicity of M78I was one of the reasons why AcrB M78I had lower susceptibility to fluoroquinolones. Fluorescence labeling experiments suggested that the AcrB M78I substitution enhanced the binding of substrates to certain amino acid sites in the efflux pathway (e.g., site Q89, E673 and F617) and weakened the binding to other amino acids (e.g., S134 and N274). Structural modeling disclosed the increased flexibility of Leu was favorable for the functional rotation of AcrB compared to the original Pro. AcrA 319L makes the functional rotation of AcrB more flexible, this enables substrate efflux more efficiently. In order to understand the mechanism of AcrAB-TolC drug efflux well, interaction between AcrA and AcrB in the role of substrate efflux of AcrAB-TolC should be further investigated.

Extracellular Histones Trigger Disseminated Intravascular Coagulation by Lytic Cell Death.

Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.

Application of Fluorescence in Studying Therapeutic Enzymes.

Fluorescence spectroscopy is one of the most important techniques in the study of therapeutic enzymes. The fluorescence phenomenon has been discovered and exploited for centuries, while therapeutic enzymes have been used in treatment of disease for only decades. This chapter provides a brief summary of the current applications of fluorescence methods in studying therapeutic enzymes to provide some insights on the selection of proper method tailored to the goal. First a brief introduction about therapeutic enzymes and history of fluorescence were provided, followed by discussions on how fluorescence was applied in the studies. Four popular fluorescence methods are discussed: fluorescence tracing, fluorescence resonance energy transfer (FRET), fluorescence quenching and fluorescence polarization. Selected application of the fluorescence methods in studying therapeutic enzymes are listed, and discussed in details in the following paragraphs.

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease.

The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.

Characterization of an acetohydroxy acid synthase mutant conferring tolerance to imidazolinone herbicides in rice (Oryza sativa).

MAIN CONCLUSION: The acetohydroxy acid synthase S627N mutation confers herbicide tolerance in rice, and the rice variety containing this mutation produces good yields. This variety is commercially viable at Shanghai and Jiangsu regions in China. Weedy rice is a type of rice that produces lower yields and poorer quality grains than cultivated rice. It plagues commercial rice fields in many countries. One strategy to control its proliferation is to develop rice varieties that are tolerant to specific herbicides. Acetohydroxy acid synthase (AHAS) mutations have been found to confer herbicide tolerance to rice. Here, we identified a single mutation (S627N) in AHAS from an indica rice variety that conferred tolerance against imidazolinone herbicides, including imazethapyr and imazamox. A japonica rice variety (JD164) was developed to obtain herbicide tolerance by introducing the mutated indica ahas gene. Imidazolinone application was sufficient to efficiently control weedy rice in the JD164 field. Although the imazethapyr treatment caused dwarfing in the JD164 plants, it did not significantly reduce yields. To determine whether the decrease of the ahas mRNA expression caused the dwarfism of JD164 after imazethapyr application, we detected the ahas mRNA level in plants. The abundance of the ahas mRNA in JD164 increased after imidazolinone application, thus excluding the mRNA expression level as a possible cause of dwarfism. Activity assays showed that the mutated AHAS was tolerant to imidazolinone but the catalytic efficiency of the mutated AHAS decreased in its presence. Moreover, the activity of the mutated AHAS decreased more in the presence of imazethapyr than in the presence of imazamox. We observed no difference in the AHAS secondary structures, but homology modeling suggested that the S627N mutation enabled the substrate to access the active site channel in AHAS, resulting in imidazolinone tolerance. Our work combined herbicides with a rice variety to control weedy rice and showed the mechanism of herbicide tolerance in this rice variety.

The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein.

ATP-dependent degradation plays a critical role in the quality control and recycling of proteins in cells. However, complete degradation of membrane proteins by ATP-dependent proteases in bacteria is not well-studied. We discovered that the degradation of a multidomain and multispan integral membrane protein AcrB could be facilitated by the introduction of a ssrA-tag at the C-terminus of the protein sequence and demonstrated that the cytoplasmic unfoldase-protease complex ClpXP was involved in the degradation. This is the first report to our knowledge to reveal that the ClpXP complex is capable of degrading integral membrane proteins. The chaperone SspB also played a role in the degradation. Using purified proteins, we demonstrated that the addition of the ssrA-tag did not drastically affect the structure of AcrB, and the degradation of detergent solubilized AcrB by purified ClpXP could be observed in vitro.

Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method.

Proteins are constantly synthesized and degraded in living cells during their growth and division, often in response to metabolic and environmental conditions. The synthesis and breakdown of proteins under different conditions reveal information about their mechanism of function. The metabolic incorporation of non-natural amino acid azidohomoalanine (AHA) and subsequent labeling via click chemistry emerged as a non-radioactive strategy useful in the determination of protein kinetics and turnover. We used the method to monitor the degradation of two proteins involved in the multidrug efflux in Escherichia coli, the inner membrane transporter AcrB and its functional partner membrane fusion protein AcrA. Together they form a functional complex with an outer membrane channel TolC to actively transport various small molecule compounds out of E. coli cells. We found that both AcrA and AcrB lasted for approximately 6 days in live E. coli cells, and the stability of AcrB depended on the presence of AcrA but not on active efflux. These results lead to new insight into the multidrug resistance in Gram-negative bacteria conferred by efflux.

Structure of inorganic pyrophosphatase from Staphylococcus aureus reveals conformational flexibility of the active site.

Cytoplasmic inorganic pyrophosphatase (PPiase) is an enzyme essential for survival of organisms, from bacteria to human. PPiases are divided into two structurally distinct families: family I PPiases are Mg(2+)-dependent and present in most archaea, eukaryotes and prokaryotes, whereas the relatively less understood family II PPiases are Mn(2+)-dependent and present only in some archaea, bacteria and primitive eukaryotes. Staphylococcus aureus (SA), a dangerous pathogen and a frequent cause of hospital infections, contains a family II PPiase (PpaC), which is an attractive potential target for development of novel antibacterial agents. We determined a crystal structure of SA PpaC in complex with catalytic Mn(2+) at 2.1Å resolution. The active site contains two catalytic Mn(2+) binding sites, each half-occupied, reconciling the previously observed 1:1 Mn(2+):enzyme stoichiometry with the presence of two divalent metal ion sites in the apo-enzyme. Unexpectedly, despite the absence of the substrate or products in the active site, the two domains of SA PpaC form a closed active site, a conformation observed in structures of other family II PPiases only in complex with substrate or product mimics. A region spanning residues 295-298, which contains a conserved substrate binding RKK motif, is flipped out of the active site, an unprecedented conformation for a PPiase. Because the mutant of Arg295 to an alanine is devoid of activity, this loop likely undergoes an induced-fit conformational change upon substrate binding and product dissociation. This closed conformation of SA PPiase may serve as an attractive target for rational design of inhibitors of this enzyme.

Cysteine residue is not essential for CPM protein thermal-stability assay.

A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

Correlation between AcrB trimer association affinity and efflux activity.

The majority of membrane proteins function as oligomers. However, it remains largely unclear how the oligomer stability of protein complexes correlates with their function. Understanding the relationship between oligomer stability and activity is essential to protein research and to virtually all cellular processes that depend on the function of protein complexes. Proteins make lasting or transient interactions as they perform their functions. Obligate oligomeric proteins exist and function exclusively at a specific oligomeric state. Although oligomerization is clearly critical for such proteins to function, a direct correlation between oligomer affinity and biological activity has not yet been reported. Here, we used an obligate trimeric membrane transporter protein, AcrB, as a model to investigate the correlation between its relative trimer affinity and efflux activity. AcrB is a component of the major multidrug efflux system in Escherichia coli. We created six AcrB constructs with mutations at the transmembrane intersubunit interface, and we determined their activities using both a drug susceptibility assay and an ethidium bromide accumulation assay. The relative trimer affinities of these mutants in detergent micelles were obtained using blue native polyacrylamide gel electrophoresis. A correlation between the relative trimer affinity and substrate efflux activity was observed, in which a threshold trimer stability was required to maintain efflux activity. The trimer affinity of the wild-type protein was approximately 3 kcal/mol more stable than the threshold value. Once the threshold was reached, an additional increase of stability in the range observed had no observable effect on protein activity.

Insights into the function and structural flexibility of the periplasmic molecular chaperone SurA.

SurA is the primary periplasmic molecular chaperone that facilitates the folding and assembling of outer membrane proteins (OMPs) in Gram-negative bacteria. Deletion of the surA gene in Escherichia coli leads to a decrease in outer membrane density and an increase in bacterial drug susceptibility. Here, we conducted mutational studies on SurA to identify residues that are critical for function. One mutant, SurA(V37G), significantly reduced the activity of SurA. Further characterization indicated that SurA(V37G) was structurally similar to, but less stable than, the wild-type protein. The loss of activity in SurA(V37G) could be restored through the introduction of a pair of Cys residues and the subsequent formation of a disulfide bond. Inspired by this success, we created three additional SurA constructs, each containing a disulfide bond at different regions of the protein between two rigid secondary structural elements. The formation of disulfide bond in these mutants has no observable detrimental effect on protein activity, indicating that SurA does not undergo large-scale conformational change while performing its function.

Role of a conserved residue R780 in Escherichia coli multidrug transporter AcrB.

Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.

Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection.

Inflammasome activation is a critical defense mechanism against bacterial infection. Previous studies suggest that inflammasome activation protects against Salmonella oral infection. Here we find inflammasome activation plays a critical role in the pathogenesis of Salmonella systemic infection. We show that in a systemic infection model by i.p. injection of Salmonella, deficiency of caspase-1 or gasdermin-D prolonged survival time, reduced plasma concentrations of the proinflammatory cytokines IL-1ß, IL-6 and TNFα. These deficiencies also protected against coagulopathy during Salmonella infection as evidenced by diminished prolongation of prothrombin time and increase in plasma thrombin-antithrombin complex concentrations in the caspase-1 or gasdermin-D deficient mice. Activation of the NAIP/NLRC4 inflammasome by flagellin and/or the components of the SPI1 type 3 secretion system played a critical role in Salmonella-induced coagulopathy. In the absence of flagellin and SPI1, the Salmonella mutant strain still triggered coagulopathy through the caspase-11/NLRP3 pathway. Our results reveal a previously undisclosed role of the inflammasomes and pyroptosis in the pathogenesis of Salmonella systemic infection.

Inhibiting NINJ1-dependent plasma membrane rupture protects against inflammasome-induced blood coagulation and inflammation.

Systemic blood coagulation accompanies inflammation during severe infection like sepsis and COVID. We've previously established a link between pyroptosis, a vital defense mechanism against infection, and coagulopathy. During pyroptosis, the formation of gasdermin-D (GSDMD) pores on the plasma membrane leads to the release of tissue factor (TF)-positive microvesicles (MVs) that are procoagulant. Mice lacking GSDMD release fewer TF MVs. However, the specific mechanisms leading from activation of GSDMD to MV release remain unclear. Plasma membrane rupture (PMR) in pyroptosis was recently reported to be actively mediated by the transmembrane protein Ninjurin-1 (NINJ1). Here we show that NINJ1 promotes procoagulant MV release during pyroptosis. Haploinsuffciency or glycine inhibition of NINJ1 limited the release of procoagulant MVs and inflammatory cytokines and protected against blood coagulation and lethality triggered by bacterial flagellin. Our findings suggest a crucial role for NINJ1-dependent PMR in inflammasome-induced blood coagulation and inflammation.

A monoadduct generating Ru(ii) complex induces ribosome biogenesis stress and is a molecular mimic of phenanthriplatin.

Ruthenium complexes are often investigated as potential replacements for platinum-based chemotherapeutics in hopes of identifying systems with improved tolerability in vivo and reduced susceptibility to cellular resistance mechanisms. Inspired by phenanthriplatin, a non-traditional platinum agent that contains only one labile ligand, monofunctional ruthenium polypyridyl agents have been developed, but until now, few demonstrated promising anticancer activity. Here we introduce a potent new scaffold, based on [Ru(tpy)(dip)Cl]Cl (tpy = 2,2':6',2''-terpyridine and dip = 4,7-diphenyl-1,10-phenanthroline) in pursuit of effective Ru(ii)-based monofunctional agents. Notably, the extension of the terpyridine at the 4' position with an aromatic ring resulted in a molecule that was cytotoxic in several cancer cell lines with sub-micromolar IC50 values, induced ribosome biogenesis stress, and exhibited minimal zebrafish embryo toxicity. This study demonstrates the successful design of a Ru(ii) agent that mimics many of the biological effects and phenotypes seen with phenanthriplatin, despite numerous differences in both the ligands and metal center structure.

Differentiating surface and bulk interactions using localized surface plasmon resonances of gold nanorods.

We demonstrate a novel localized surface-plasmon resonance sensor that can distinguish surface binding interactions from interfering bulk effects. This is accomplished by utilizing the longitudinal and transverse plasmon modes of gold nanorods. We have investigated, both numerically and experimentally, the effect of change in background refractive index and surface binding on the two resonances of a gold nanorod on an indium tin oxide coated glass substrate.

Oriented immobilization of proteins on hydroxyapatite surface using bifunctional bisphosphonates as linkers.

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and ß-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 × 10(-5) mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10-15 fold that of adsorbed EGFP, whereas the relative amount of ß-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed ß-lactamase. The enzymatic activity of HBP-immobilized ß-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized ß-lactamase improved 2-5 fold over adsorbed ß-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.

Accumulation and efflux of polychlorinated biphenyls in Escherichia coli.

Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB-TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB-TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria.

Transport Across Two Membrane Bilayers in E. coli by Efflux Pumps of Different Dimensions.

AcrAB-TolC and CusBAC are two of the most well-studied Resistance-Nodulation-Division (RND) family tripartite efflux pumps in E. coli. AcrAB-TolC is a multidrug efflux system, while CusBAC transports Cu(I), Cu(II) and Ag(I). The RND pump complexes span both the inner membrane (IM) and the outer membrane (OM). The long axis dimension of the fully assembled AcrAB-TolC is ∼3 nm longer than that of CusBAC. To probe if these two efflux systems with different dimensions affect each other when they need to work simultaneously in the same cell, two real-time assays were used to monitor the efflux activities of these two pumps and their impact on each other. The results showed that the presence of AcrAB-TolC substrates accelerated the accumulation of Cu(I) in BW25113 but not in BW25113ΔcusBA or BW25113ΔtolC strains. Similarly, the presence of Ag(I) slowed down the Nile red efflux in the parent strain more significantly than in the CusBA deficient mutant. To further investigate the impact of the OM/IM distance on the function of these tripartite complexes, we experimented with strains lacking the lipoprotein Lpp or containing Lpp mutant of different lengths. Data from efflux/accumulation assays and susceptibility tests revealed that mutation of Lpp resulted in functional deficiency of both AcrAB-TolC and CusBAC. In conclusion, this study demonstrated that when AcrAB-TolC and CusBAC functioned simultaneously, it took the cell a few minutes to adjust. Furthermore, the presence of Lpp of proper length is important to support full efflux activity of transporters spanning both membrane layers in E. coli.